Henseleit

In 1937 Krebs found that citrate could be formed in muscle suspensions if oxaloacetate and either pyruvate or acetate were added. He saw that he now had a cycle, not a simple pathway, and that addition of any of the intermediates could generate all of the others. The existence of a cycle, together with the entry of pyruvate into the cycle in the synthesis of citrate, provided a clear explanation for the accelerating properties of succinate, fumarate, and malate. If all these intermediates led to oxaloacetate, which combined with pyruvate from glycolysis, they could stimulate the oxidation of many substances besides themselves. (Kreb s conceptual leap to a cycle was not his first. Together with medical student Kurt Henseleit, he had already elucidated the details of the urea cycle in 1932.) The complete tricarboxylic acid (Krebs) cycle, as it is now understood, is shown in Figure 20.4. 

FIG. 2. Effects of LSD on the spontaneous and K+-induced release of newly synthesized 3H-5-HT in brainstem slices of 12-hr-, 7-day-, or 90-day-old rats. At the end of a 30 min incubation with 3H-tryptophan (0.56 mm) under conditions indicated in the abscissa, 3H-5-HT was measured separately in tissues and medium. The ratio (3H-5-HT%) of 3H-5-HT found in medium over that in tissues is expressed in percentage of that calculated for brainstem slices incubated in normal Krebs-Henseleit medium (K+ = 5.6 mu) in the absence of LSD. Bar, mean SEM of six to eight separate determinations. p < 0.05 when compared with control values (K+ = 5.6 mM, no LSD) p < 0.05 when compared with values found with slices incubated in K+-enriched medium in the absence of LSD. 

The first suggestion that substrates in carbohydrate oxidation might exert catalytic effects on the oxidation of other intermediates (cf.earlier demonstration of such action in the urea cycle by Krebs and Henseleit, 1932 see Chapter 6) arose from the work of Szent-Gyorgi (1936). He demonstrated that succinate and its 4C oxidation products catalytically stimulated the rate of respiration by muscle tissues. He also observed that reactions between the 4C intermediates were reversible and that if muscle was incubated with oxaloacetate, fumarate and malate made up 50-75% of the products, 2-oxoglutarate 10-25% and, significantly, 1-2% of the C was converted to citrate. These observations were... 

However Henseleit showed there was a correlation between the concentration of ornithine and the magnitude of the effect on urea production. That the absolute amounts of ornithine were so small provided Krebs with arguments for the idea that the ornithine acted catalytically. 

The paper by Krebs and Henseleit (1932), Experiments on the Formation of Urea in Animal Bodies (Klinische Wochenschrift 11,759), contained the phrases... in the synthesis of urea in the living cell, ornithine acts like a catalyst. We therefore draw the conclusion. .. that the primary reaction for the synthesis of urea from ammonia is... 

Lohman discovered ATP in muscles. Krebs and Henseleit. The urea cycle. Svedberg began studies with the ultracentrifuge. 

On an average Western diet, adnlt hnmans excrete around 30 g of nrea per day but this can easily triple on a protein-rich diet. The reactions and the concept of a cycle were discovered by Krebs Henseleit (1932). Snbseqnent work clari-hed the details of what has become known as the ornithine or the nrea cycle. 

The cores are subsequently placed in the sheer, and the slicing procedure is performed by advancing the core over an oscillating knife in a controlled environment (Figure 12.1). Cold (4°C) Krebs-Henseleit buffer (pH = 7.4, saturated with 95% O2 and 5% CO2) supplemented with 25 mM glucose is commonly used in preparing the slices [35,38 0], but Williams medium E [41], Earle s balanced salt solutions [37], Sacks preservation medium [42] and V-7 preservation buffer [43,44] are also used. 

In ureotelic organisms, the ammonia deposited in the mitochondria of hepatocytes is converted to urea in the urea cycle. This pathway was discovered in 1932 by Hans Krebs (who later also discovered the citric acid cycle) and a medical student associate, Kurt Henseleit. Urea production occurs almost exclusively in the liver and is the fate of most of the ammonia channeled there. The urea passes into the bloodstream and thus to the kidneys and is excreted into the urine. The production of urea now becomes the focus of our discussion. 

Urea formation in the liver starts with the multistep conversion of ornithine to arginine (fig. 22.7). This is followed by the breakdown of arginine into ornithine and urea. The cyclic nature of this pathway was first appreciated by Hans Krebs and Henseleit in 1932. In subsequent years the impor-... 

Dog No. 3 was omitted because of insufficient data. It is apparent that a marked uptake of ammonia occurs in tissues in metabolic acidosis. In alkalosis the uptake by tissues is only slightly greater than normal, and the arterial levels of ammonia are comparable with normal values. The elevation of blood ammonia which occurs on vigorous acidification of the animal might not be due to a drop in permeability with pH, but might be due to a phenomenon reported by Krebs and Henseleit (K4). Urea synthesis by liver slices is directly proportional to the pH and C02 content of the medium. Table 2, constructed from data reported in this paper, shows this dependence. 

The urea cycle Urea is synthesized in the liver by the urea cycle. It is then secreted into the bloodstream and taken up by the kidneys for excretion in the urine. The urea cycle was the first cyclic metabolic pathway to be discovered by Hans Krebs and Kurt Henseleit in 1932,5 years before Krebs discovered the citric acid cycle (see Topic LI). The overall reaction of the pathway is ... 

Typical experimental procedures are as follows The test drug candidate is incubated with pooled human liver microsomes (e.g., 1 mg protein/mL) that were previously preincubated with ABT (1 or 2 mM) for 30 minutes at (37 1)°C in the presence of an NADPH-generating system. Incubations of the drug candidate in the absence of ABT serve as controls. For hepatocytes, suspensions of freshly isolated or cryopreserved hepatocytes (lx 106 cells/ mL) are preincubated with 100-pM ABT for 30 minutes in 0.25 mL of Krebs-Henseleit buffer or Waymouth s medium (without phenol red) supplemented with FBS (4.5%), insulin (5.6 pg/mL), glutamine (3.6 mM), sodium pyruvate (4.5 mM), and dexamethasone (0.9 pM) at the final concentrations indicated. After the preincubation, the drug candidate is added to the incubation and the rate of metabolism of the drug candidate is compared in hepatocytes or microsomes with and without ABT treatment. A marked difference in metabolism caused by ABT is evidence that CYP plays a prominent role in the metabolism of the drug candidate. 

Production of urea by cestodes suggests the existence of the urea (Krebs-Henseleit) cycle, which is shown in Fig. 6.11. One of the key enzymes, arginase, has been widely reported in cestodes (796, 185-187). However, some of the other enzymes, notably carbamyl phosphate synthetase and ornithine transcarbamyl, are either absent or present in only low amounts (39) and it is doubtful if a complete cycle operates in cestodes. It is likely that the urea excreted by tapeworms comes from the activity of arginase alone. The uric acid produced and excreted by cestodes probably arises from the breakdown of purines (39). 

Fig. 6.11. The (Krebs-Henseleit) ornithine cycle. Numbers refer to enzymes as follows. (1) Carbamyl phosphate synthetase (E.C.2.7.2.a). (2) Ornithine transcarbamylase (E.C.2.1.3.3). (3) Arginino-succinate synthetase (E.C.6.3.4.5). (4) Arginino-succinate lyase (E.C.4.3.2.1). (5) Arginase (E.C.3.5.3.1). (After Smyth, 1969.)...

Urea is synthesized via the urea cycle (Fig. 18-1). In 1932, Krebs and Henseleit pubEshed data demonstrating that ornithine stimulates the synthesis of urea without stoichiometric consumption of this intermediate. This apparent catalytic function was determined to be the result of the cycEc nature of the pathway. This was a revolutionary idea since metabolic pathways were conceptualized as purely linear prior to the pubEcation of these observations. In the foUowing sections, we discuss the biochemical processes involved in urea formation. 

After the equilibration period, clearance periods of 20 min are used. Urine samples are collected and perfusate is obtained at midpoint of the clearance period for the evaluation of overall kidney function. For determination of glomerular filtration rate (GFR) and fluid transport, 3H-labelled polyethylene glycol is added to a modified Krebs-Henseleit bicarbonate buffer. Electrolytes are determined in urine by standard flame photometry. Fractional excretions of water, electrolytes and test compounds are calculated. 

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