Harry Goldblatt

Extraction, Purification, and Assay of Human Renin Free of Angiotensinase

Three procedures are described for the extraction and purification of renin from 0.05 to 500 g of human kidneys. A uniform yield of renin, free of angiotensinase, resulted from all three procedures. All tests for renin were carried out in dogs. When more than 10 g of renal tissue was used, renin was determined by the direct method; with smaller amounts, the indirect method, involving the production and the assay of angiotensin, was employed. Renin substrate free of angiotensinase, suitable for the indirect assay of human renin, was prepared from pooled human serum by a simple procedure. The angiotensinase-free renin and substrate permitted prolonged incubation for the production of the angiotensin required for the indirect assay. The mean ratio of angiotensin produced (unit per milliter of serum) to the amount of renin added (unit per milliliter of serum) was 1,482 for an 18-hour period of incubation. The large amount of angiotensin produced permitted the indirect assay of minute amounts of human renin (as little as 0.0005 unit, from 5 mg of renal tissue) in the dog. Unless the angiotensinase present in extracts of renal tissue or serum is first removed, the accurate, indirect assay of the renin is not possible.

Extraction, purification, and acetylation of human renin and the production of antirenin to human renin.

Abstract Bioassay procedures employed for the quantitative determination of human renin; antirenin to human renin; angiotensin; and angiotensinase are described. All of these assays are based on the determination of the increase of the direct, mean systemic blood pressure of trained, unanesthetized dogs after intravenous injection of the test substances. Four preparations of commercially available angiotensin II (Hypertensin Ciba) were assayed and found to contain 2000–2200 dog units of angiotensin per milligram. The extraction and purification of renin were carried out on batches up to 35 kg of human kidneys. This resulted in an over-all yield of 60% and a 600-fold purification of the renin, including the complete removal of angiotensinase. The immunization of rats, rabbits, and a dog with human renin resulted in the production of antirenin to human renin with maximum titers of 7, 12, and 11 units per milliliter of serum, respectively. The acetylation of human renin induced a decrease of its enzymic (pressor) activity and an alteration of its antigenic structure, but not an impairment of its antigenicity. As a result, the immunization of a dog with acetylated human renin produced antirenin of a high titer, with cross-reactivity toward both untreated and acetylated human renin. In its chemical and immunological characteristics, human renin differs from that of other species: (a) Human renin was not acetylated under conditions found to be effective for renin of five other species; it required different conditions, such as a higher temperature and a more alkaline reaction. (b) Antirenin which had been produced with untreated human renin, or with acetylated human renin, failed to react with the renin of hog, dog, rabbit, beef, sheep, and rat. The acetylation of the renin preparation obtained after Step 8 of the new isolation procedure lowered the pressor activity to 36% and the amount of amino groups to approximately 24% of the original value.

ESTIMATION OF ENDOGENOUS RENIN IN HUMAN BLOOD

Abstract With existing methods, estimates of endogenous renin in human serum or plasma differ widely, in part due to technical differences. In a new relatively simple and less time-consuming method the essential technical details are the use of purified, angiotensinase-free substrate added to angiotensinase-free serum, incubation for a prolonged period, and control assays of increasing quantities of a standard preparation of human renin added to the serum. Under these conditions, the yield of angiotensin increases linearly with the concentration of the added renin, and the concentration of endogenous renin in the serum can be calculated from the slope of the line, which represents the quantity of angiotensin produced per unit of added renin, and the intercept, which represents the amount of angiotensin produced without added renin. A precise assay of renin expressed in terms of an internationally acceptable standard preparation of human renin can thus be achieved.

Kinetic constants of the human renin and human angiotensinogen reaction.

The rate of formation of angiotensin was investigated for the reaction of angiotensinase-free human renin, at various states of purity, with angiotensinogen, in pooled, angiotensinase-free human serum. The velocity constant of this reaction and the Michaelis constant of the renin-substrate complex were determined. The concentration of angiotensinogen and of endogenous renin in human serum was also determined and, on the basis of these results, a procedure was designed for the indirect assay of human renin. This was carried out under a wide range of experimental conditions, for periods of incubation ranging from 10 min to 18 hours, for various concentrations of substrate, and for renin concentrations varying from 0.000025 to 0.20 unit/ml. The absence of angiotensinase made possible the prolonged incubation (18 hours) of a minute quantity of renin with a large amount of the renin substrate. The resultant formation of large amounts of angiotensin permitted its accurate assay in the dog.

Studies on Renin I. PURIFICATION OF DOG RENIN AND MICROMETHOD FOR THE DETERMINATION OF RENIN IN DOG SERUM AND TISSUES

Dog renin was produced with a yield of 1.1 Goldblatt units (GU)/g renal tissue and with a specific activity of 1.3 GU/mg protein, i.e., a specific activity 10-16 times higher than that of previous preparations. This renin, free of angiotensinase and of anaphylactoid substances, was used to study the effects of prolonged infusion of renin in normal conscious dogs and as a standard for the bioassay of renin. A simplified method was designed for the indirect assay of canine renin. After prolonged incubation, the angiotensin produced was determined by bioassay in the rat, and the final estimate was expressed as the concentration of renin, in terms of GU, with an accuracy of ± 12.8%. The lower sensitivity of the assay is 1.0 x 10-5 GU/ml; this limit permits an estimate of the endogenous renin in 0.1 ml of normal dog serum or in 0.005 mg of normal canine renal tissue. The sensitivity of this bioassay is, therefore, within the range found in seven radioimmunoassays (0.26 x 10-5 GU/ml to 6.2 x 10-5 GU/ml), and co...

On the malignant transformation of cells during prolonged culture under hypoxic conditions in vitro

Four independent series of mixed cultures of epithelial cells and fibroblasts, from the skin (mainly epidermis) of rat embryos, grown under a layer of fluid medium in stationary T-60 flasks, with or without free access of ambient air to the surface of the medium, eventually yielded malignant tumors, when the cultures were injected into rats of the same strain. These cultures were composed of both types of cell, and proved transplantable when injected subcutaneously or intraperitoneally, even into untreated rats of the same strain as the embryos. Exposure of the cells to a high concentration of readily available oxygen in the medium resulted in at least a delay, and probably the complete prevention, of the malignant transformation of the cells. Observations of the malignant transformation of cells grown for prolonged periods in vitro have become commonplace, but the cause of the phenomenon has remained obscure. Neither a virus nor any known physical or chemical carcinogenic agent has been proved to be the cause. The transformation of the cells was not reversed during 13 months of exposure of the cultures of malignant cells to a high concentration of oxygen in the medium. The experimental results of this investigation, as well as the theoretical considerations, suggest that intracellular hypoxia, due to the inadequate rate of diffusion of the oxygen in the ambient air through the stationary layer of liquid medium, acted at least as the initiating agent, with unknown (intrinsic or extrinsic) factors possibly acting as the promoting carcinogenic agent.

Studies on Renin: II. CONTINUOUS INFUSION OF HOMOLOGOUS RENIN AT VERY LOW RATES IN INTACT OR NEPHRECTOMIZED, CONSCIOUS OR ANESTHETIZED DOGS

We have devised a method of evaluation, in terms of Goldblatt units (GU), of the data obtained in various laboratories for the excretion of renin from canine and human kidneys. An average value of 1.3 × 10−4 GU/min g−1 normal renal tissue resulted for both dog and man. A blood pressure increase of 18−45 mm Hg was induced in normal or nephrectomized, conscious or anesthetized dogs by the continuous infusion of homologous renin at even lower rates (0.25−0.50 × 10−4 GU/min g−1 renal tissue), i.e., at 20−40% of the rate excreted by the kidney. This suggests that renin, even at its normal rate of excretion from the kidney, participates in the maintenance of normal blood pressure. The increase of blood pressure and of concentration of renin in the dogs blood was measured at infusion rates (C) of 0.0042, 0.0083, 0.0167, 0.0333, and 0.0667 GU/min. At each rate, after a fixed period of infusion (t = 60 minutes), a constant fraction (54% of the infused renin) was retained in the circulation. From this and the results of infusion experiments carried out for 30, 60, and 120 minutes, the total amount of exogenous renin, R, present in the circulation at time t can be expressed as R = C/K (1 − e−Kt). K = 0.0232 minutes−1 and represents the fraction of exogenous and, presumably, also endogenous renin that is being eliminated from the circulation of the normal dog per minute. Thus, the time required for removal of 50% of the renin from the blood is t½ = loge 2/K = 30 minutes.

Direct percutaneous determination of systemic blood pressure and production of renal hypertension in the cat.

Summary The percutaneous, direct method for determination of the blood pressure was applied in the cat, without and with anesthesia. In one group of 5 males and 5 females, single determinations were made, to determine the feasibility of the method. In these animals the blood pressure was measured by 2 means—direct mean pressure with a mercury manometer, and systolic and dia-stolic pressures with a strain-gauge manometer. In this group the single pressures were generally high, and usually higher in the males. In another group, 2 males and 2 females, determinations were made weekly for 7 to 24 weeks. In these cats the initial pressures were also high, but similar in both sexes, but, in all the animals, gradually decreased and became stabilized at a lower level in about 4 weeks. In one male and in one female the production of unilateral renal ischemia, due to moderate constriction of the main renal artery, was followed by the development of hypertension, and, as in other species, the exclusion of the ischemic kidney of one of the hypertensive cats resulted in a prompt return of the blood pressure to the lowest prehypertensive level.