Development and evaluation of a pan-dermatophyte polymerase chain reaction with species-level identification using sloppy molecular beacon probes.

BACKGROUND: Conventional laboratory diagnosis of dermatophyte infection is cumbersome and time-consuming. OBJECTIVES: We aimed to establish a simple, robust and rapid molecular diagnostic assay for the detection of dermatophytes and optionally nondermatophytes in clinical specimens. MATERIALS AND METHODS: We developed a two-tube pan-dermatophyte polymerase chain reaction (PCR) assay using six sloppy molecular beacon (SMB) probes. The first PCR uses dermatophyte-specific primers and enables detection and identification of most dermatophyte species. The second PCR with pan-fungal primers allows further differentiation of Trichophyton interdigitale and T. mentagrophytes/T. quinckeanum, T. violaceum and T. soudanense, and T. tonsurans and T. equinum, and detection of nondermatophytes. The test was evaluated with 306 clinical specimens by comparing it with the results of microscopy and culture. RESULTS: In melting-curve analyses, species-specific melting temperature signatures of the SMBs were defined, and thus, our new PCR enabled detection and species-level identification of at least 19 dermatophyte species. Sensitivity and specificity of PCR for detection of dermatophytes in clinical samples were estimated to be 96·9% and 90·4%, for culture 46·7% and 98·7%, and for microscopy 91·4% and 84·0%, respectively. The detection of nondermatophytes by PCR and culture did not correlate. CONCLUSIONS: The new assay showed excellent performance characteristics for the detection of dermatophytes and is significantly faster than culturing techniques, which makes it very promising for routine diagnostics of dermatophytosis. We noted that the detection of nondermatophytes in our assay currently has no benefit.

Clinical Outcome of Sacral Chordoma Patients Treated with Pencil Beam Scanning Proton Therapy.

AIMS: Sacral chordomas are locally aggressive, radio-resistant tumours. Proton therapy has the potential to deliver high radiation doses, which may improve the therapeutic ratio when compared with conventional radiotherapy. We assessed tumour control and radiation-induced toxicity in a cohort of sacral chordoma patients treated with definitive or postoperative pencil beam scanning proton therapy. METHODS AND MATERIALS: Sixty patients with histologically proven sacral chordoma treated between November 1997 and October 2018 at the Paul Scherrer Institute with postoperative (n = 50) or definitive proton therapy (n = 10) were retrospectively analysed. Only 10 (17%) patients received combined photon radiotherapy and proton therapy. Survival rates were calculated using the Kaplan-Meier actuarial method. The Log-rank test was used to compare different functions for local control, freedom from distant recurrence and overall survival. Acute and late toxicity were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) v5.0. RESULTS: The median follow-up was 48 months (range 4-186). Local recurrence occurred in 20 (33%) patients. The 4-year local control, freedom from distant recurrence and overall survival rates were 77%, 89% and 85%, respectively. On univariate analysis, subtotal resection/biopsy (P = 0.02), tumour extension restricted to bone (P = 0.01) and gross tumour volume >130 ml (P = 0.04) were significant predictors for local recurrence. On multivariate analysis, tumour extension restricted to bone (P = 0.004) and gross total resection (P = 0.02) remained independent favourable prognostic factors for local recurrence. Twenty-four (40%), 28 (47%) and eight (11%) patients experienced acute grade 1, 2 and 3 toxicities, respectively. The 4-year late toxicity-free survival was 91%. Two patients developed secondary malignancies to the bladder 3-7 years after proton therapy. CONCLUSIONS: Our data indicate that pencil beam scanning proton therapy for sacral chordomas is both safe and effective. Gross total resection, tumour volume <130 ml and tumour restricted to the bone are favourable prognostic factors for local tumour control.

Assessing the advantages of CFR-PEEK over titanium spinal stabilization implants in proton therapy-a phantom study.

High-density materials, such as titanium, used for spinal stabilization, introduces several critical issues in proton therapy (PT). Artefacts affect both contouring and dose calculation. Subsequently, artefacts need to be corrected which is a time-consuming process. Besides, titanium causes proton interactions that are unaccounted for in dose calculation. The result is a suboptimal treatment plan, and indeed decreased local controls have been reported for these patients. Carbon fiber reinforced polyetheretherketone (CFR-PEEK) implant material, which is of low density, potentially solves these issues. For this study, we designed a unique phantom to compare the effects of titanium and CFR-PEEK implants in PT. The phantom contains four interchangeable spinal inserts representing a native spine, and three different spinal stabilizations consisting of titanium only, CFR-PEEK only, and a combination of titanium and CFR-PEEK. All phantom scenarios received the standard treatment workup. Two planning approaches were investigated: a single field plan and a multi-field optimized plan with spinal cord sparing. For both plans we analyzed the following aspects: total volume of artefacts on CT images, time required for artefact correction, effect of planning CT correction on dose calculation, plan robustness to range and set up uncertainties, and finally the discrepancy between the calculated dose and the delivered dose with Gafchromic® film. The CFR-PEEK implant had a 90% reduction of artefacts on CT images and subsequently severely reduced the time for artefact correction with respect to the titanium-only implant. Furthermore, the CFR-PEEK as opposed to titanium did not influence the robustness of the plan. Finally, the titanium implants led to hardware-related discrepancies between the planned and the measured dose while the CFR-PEEK implant showed good agreement. As opposed to titanium, CFR-PEEK has none to minor effects on PT. The use of CFR-PEEK is expected to optimize treatment and possibly improve outcomes for patients that require spinal stabilization.

Pencil Beam Scanning Proton Therapy for Paediatric Neuroblastoma with Motion Mitigation Strategy for Moving Target Volumes.

AIMS: More efforts are required to minimise late radiation side-effects for paediatric patients. Pencil beam scanning proton beam therapy (PBS-PT) allows increased sparing of normal tissues while maintaining conformality, but is prone to dose degradation from interplay effects due to respiratory motion. We report our clinical experience of motion mitigation with volumetric rescanning (vRSC) and outcomes of children with neuroblastoma. MATERIALS AND METHODS: Nineteen patients with high-risk (n = 16) and intermediate-risk (n = 3) neuroblastoma received PBS-PT. The median age at PBS-PT was 3.5 years (range 1.2-8.6) and the median PBS-PT dose was 21 Gy (relative biological effectiveness). Most children (89%) were treated under general anaesthesia. Seven patients (37%) underwent four-dimensional computed tomography for motion assessment and were treated with vRSC for motion mitigation. RESULTS: The mean result of maximum organ motion was 2.7 mm (cranial-caudal), 1.2 mm (left-right), 1.0 mm (anterior-posterior). Four anaesthetised children (21%) showing <5 mm motion had four-dimensional dose calculations (4DDC) to guide the number of vRSC. The mean deterioration or improvement to the planning target volume covered by 95% of the prescribed dose compared with static three-dimensional plans were: 4DDC no vRSC, -0.6%; 2 vRSC, +0.3%; 4 vRSC, +0.3%; and 8 vRSC, +0.1%. With a median follow-up of 14.9 months (range 2.7-49.0) there were no local recurrences. The 2-year overall survival was 94% and distant progression-free survival was 76%. Acute grade 2-4 toxicity was 11%. During the limited follow-up time, no late toxicities were observed. CONCLUSIONS: The early outcomes of mainly high-risk patients with neuroblastoma treated with PBS-PT were excellent. With a subset of our cohort undergoing PBS-PT with vRSC we have shown that it is logistically feasible and safe. The clinical relevance of vRSC is debatable in anaesthetised children with small pre-PBS-PT motion of <5 mm.

Outcomes, Prognostic Factors and Salvage Treatment for Recurrent Chordoma After Pencil Beam Scanning Proton Therapy at the Paul Scherrer Institute.

AIMS: The outcome of chordoma patients with local or distant failure after proton therapy is not well established. We assessed the disease-specific (DSS) and overall survival of patients recurring after proton therapy and evaluated the prognostic factors affecting DSS. MATERIALS AND METHODS: A retrospective analysis was carried out of 71 recurring skull base (n = 36) and extracranial (n = 35) chordoma patients who received adjuvant proton therapy at initial presentation (n = 42; 59%) or after post-surgical recurrence (n = 29; 41%). The median proton therapy dose delivered was 74 GyRBE (range 62-76). The mean age was 55 ± 14.2 years and the male/female ratio was about one. RESULTS: The median time to first failure after proton therapy was 30.8 months (range 3-152). Most patients (n = 59; 83%) presented with locoregional failure only. There were only 12 (17%) distant failures, either with (n = 5) or without (n = 7) synchronous local failure. Eight patients (11%) received no salvage therapy for their treatment failure after proton therapy. Salvage treatments after proton therapy failure included surgery, systemic therapy and additional radiotherapy in 45 (63%), 20 (28%) and eight (11%) patients, respectively. Fifty-three patients (75%) died, most often from disease progression (47 of 53 patients; 89%). The median DSS and overall survival after failure was 3.9 (95% confidence interval 3.1-5.1) and 3.4 (95% confidence interval 2.5-4.4) years, respectively. On multivariate analysis, extracranial location and late failure (≥31 months after proton therapy) were independent favourable prognostic factors for DSS. CONCLUSION: The survival of chordoma patients after a treatment failure following proton therapy is poor, particularly for patients who relapse early or recur in the skull base. Although salvage treatment is administered to most patients with uncontrolled disease, they will ultimately die as a result of disease progression in most cases.

Gyrate atrophy of the choroid and retina: improved visual function following reduction of plasma ornithine by diet.

In a patient with gyrate atrophy of the choroid and retina, an arginine-deficient diet has reduced plasma ornithine concentration fivefold during the past 20 months. Subjective improvement in her visual function was noted approximately 15 months after institution of her diet. This has been documented by improvements in the electroretinogram, dark-adaptation, and color vision. The improvement involves rod and, to a lesser extent, cone function. The results, although preliminary and limited to a single patient, suggest that reduction of plasma ornithine with a low arginine diet is beneficial in this disease.

Urea metabolism in chronic renal failure.

Urea degradation was measured during 16 experiments in 13 chronic uremic patients being treated with essential amino acids or their analogues. [(14)C]Urea was injected i.v. and the clearance of labeled urea from its volume of distribution was compared with the simultaneous renal clearance of ordinary urea, which averaged 2.0 liters/day. The difference, extrarenal clearance of urea, averaged 3.1 liters/day as compared with a previously reported mean of 18 liters/day in normal subjects. Thus urea-splitting activity in the gut of uremic subjects expressed in these terms is far less than in normal individuals. Nevertheless, the amount of ammonia N formed from urea in these patients, 3.5 g/day, is not significantly different from normal, owing to their elevated plasma urea. In the same subjects, urea appearance rate was measured as the sum of urea excretion and the daily change in the urea pool. No negative correlation was noted between urea appearance and urea degradation, as might be expected if portal ammonia were being utilized for protein synthesis. However, urea production was positively correlated (r = 0.76) with urea degradation, suggesting that most of the resulting portal ammonia is converted back to urea. The results fail to support the view that degradation of urea in the gut promotes N conservation in uremic subjects maintained on low protein diets.

Reversible stimulation of sodium transport in the toad bladder by stretch.

Short-circuit current and transepithelial potential difference were measured in toad hemibladders mounted as sacs on glass cannulae. When sac volume was changed by adding or removing fluid, short circuit current responded by increasing or decreasing during the ensuing half-hour. The time course of the response and its magnitude indicated that it was not artefactual. Furthermore, net sodium flux responded similarly. Sac volume, and thus bladder surface area, could be varied from 0.03 to 0.4 cm(2)/mg wet weight. The mean response to either decreases or increases was 10 muA/cm(2). Everted hemibladders, however, responded less. Neither hydrostatic pressure, nor increased chloride conductance, nor increased access of oxygen or glucose to the mucosa was responsible for the response. Tissue conductance did vary markedly with volume, and may have played a role, but sodium conductance did not vary with volume in a consistent manner. The results indicate the existence of an intrinsic mechanism in this tissue which alters sodium transport in response to stretch.

Extracellular fluid in individual tissues and in whole animals: the distribution of radiosulfate and radiobromide.

Radiosulfate, (35)SO(4), and radiobromide, (82)Br, were administered simultaneously to rats and dogs. In rats, the apparent volume of distribution of (82)Br averaged 30% of body weight and was constant between 0.5 and 35 hr after injection. The apparent volume of distribution of (35)SO(4), corrected for urinary loss, increased by 6% body weight/hr: the extrapolated volume at zero time was 88% of bromide space. Analysis of individual tissues and carcasses for (82)Br and inorganic (35)SO(4) showed that equilibration of both isotopes in several organs and in the whole carcass was rapidly achieved within 1 to 2 hr: no further increase in measured spaces occurred in 24 hr. The carcass inorganic sulfate space was 92%+/-2% of the bromide space in intact rats, and showed no increase with time. However, a progressively greater fraction of the injected (35)SO(4) was not recovered, owing to metabolic alteration. In eviscerated rats, the inorganic sulfate space was a smaller and much more constant fraction (79.8% +/-0.4%) of the bromide space, showing that at least 20% of body bromide (and hence chloride) is nonextracellular. The viscera chiefly responsible for the higher ratio of spaces in the intact animal were the liver, small bowel, and kidney. In the last two organs, excess inorganic (35)SO(4) (beyond the bromide space) was attributable to trapped transcellular fluid in which sulfate had been concentrated more than chloride (or bromide). Excess sulfate in liver and cartilage could not be explained in this manner: the results suggest passive binding of sulfate, but could reflect active cell uptake in these tissues. No excess sulfate was found in skin or tail. The implications of these observations with respect to the distribution of body chloride and the measurement of extracellular space are discussed. The extracellular volume of the rat is estimated to be 24% of body weight.

Aldosterone action and sodium- and potassium-activated adenosine triphosphatase in toad bladder.

Urinary hemibladders obtained from toads soaked in water or saline were treated with aldosterone, 10(-6) M, either 1(1/2) or 16 h after mounting. After 2(1/2) h exposure to the hormone, short-circuit current was increased by 110-192% and open-circuit potential by 20-44% as compared with untreated paired hemibladders. Mucosal cells were then assayed for sodium-potassium-stimulated adenosine triphosphatase (ATPase). No increase occurred in activity per milligram protein or in the portion of total activity dependent on sodium. Activity at low sodium concentrations was also measured and analyzed by means of the Hill equation in terms of K, the apparent dissociation constant of the enzyme-sodium complex, and n, a number that expresses the degree of interaction between binding sites. Neither K nor n was significantly altered by aldosterone. A few experiments were also carried out at low ATP concentrations (0.3 mM); again no change in sodium-dependent activity was noted. The results indicate that aldosterone does not stimulate sodium transport by increasing the quantity of sodium-potassium adenosine triphosphatase in mucosal cells or the dependence of this activity on sodium or ATP concentrations.

Failure of the normal ureagenic response to amino acids in organic acid-loaded rats. Proposed mechanism for the hyperammonemia of propionic and methylmalonic acidemia.

Propionic and methylmalonic acidemia are both known to be associated with hyperammonemia. Rats injected with 10 or 20 mmol/kg of propionate or 20 mmol/kg of methylmalonate, along with 1.5 g/kg of a mixture of amino acids, developed severe hyperammonemia, whereas rats administered the same dosages of acetate did not. In vitro, neither propionyl nor methylmalonyl CoA affected the activity of carbamyl phosphate synthetase I, ornithine transcarbamylase, nor the activation constant (K(A)) of carbamyl phosphate synthetase I for N-acetyl glutamate. Furthermore, rats injected with propionate showed no alteration of liver amino acid concentrations, which could explain impaired ureagenesis. Animals injected with methylmalonate showed an increase in both citrulline and aspartate, suggesting that argininosuccinic acid synthetase may also have been inhibited. Liver ATP levels were unchanged. Citrullinogenesis, measured in intact mitochondria from livers of injected animals, was reduced 20-25% by 20 mmol/kg of propionate or methylmalonate (compared with acetate). This effect was attributable to an impairment in the normal rise of liver N-acetyl glutamate content after amino acid injection. Thus, carbamyl phosphate synthetase I activation was reduced. Liver levels of acetyl CoA and free CoA were reduced. Levels of unidentified acyl CoA derivatives rose, presumably reflecting the accumulation of propionyl and methylmalonyl CoA. Thus, the principal mechanism for hyperammonemia induced by these acids is depletion of liver N-acetyl glutamate, which is in turn attributable to depletion of acetyl CoA and/or competitive inhibition by propionyl and methylmalonyl CoA of N-acetyl glutamate synthetase. Injection of methylmalonate may also have an additional inhibitory effect on argininosuccinic acid synthetase.

Nitrogen sparing induced by leucine compared with that induced by its keto analogue, alpha-ketoisocaproate, in fasting obese man.

We measured the effects of seven consecutive daily infusions of alpha-ketoisocaproate (the alpha-keto analogue of leucine) or leucine itself on urinary urea and total nitrogen excretion during fasting. Two study protocols were undertaken. In protocol I, subjects underwent three separate 14-d fasts: one during which 34 mmol/d of leucine were infused on days 1--7; a second during which 34 mmol/d of alpha-ketoisocaproate were infused on days 1--7; and a third control fast during which no infusions were given. Infusions of alpha-ketoisocaproate significantly reduced daily urine urea nitrogen excretion compared with both the control fasts and the fasts in which leucine was infused (P less than 0.001). This nitrogen-sparing effect of alpha-ketoisocaproate persisted during days 8--14 even though no further infusions were given. Daily urinary urea nitrogen excretion during fasts when leucine was administered did not differ from values observed during control fasts. In protocol II, subjects were starved on two occasions for 14 d. During one fast, infusions of 11 mmol/d of alpha-ketoisocaproate were given on days 1--7; during the control fast, no infusions were given. Daily urine urea nitrogen excretion was lower (P less than 0.001) on days 1--7 and also on days 8--14 of the fast during which alpha-ketoisocaproate was given. The nitrogen-sparing effect of alpha-ketoisocaproate could not be related to changes in circulating levels of amino acids, ketone bodies, or insulin in either protocol. We conclude that alpha-ketoisocaproate infusions decrease the nitrogen wasting of starvation, whereas leucine, studied under identical conditions, does not.

Nitrogen sparing induced by a mixture of essential amino acids given chiefly as their keto-analogues during prolonged starvation in obese subjects.

11 normal obese subjects were fasted for 33 days. In five, who served as controls, urine urea nitrogen excretion remained constant for 2 wk thereafter. The other six were given seven daily infusions containing 6-8 mmol each of the alpha-keto-analogues of valine, leucine, isoleucine, phenylalanine, and methionine (as sodium salts) plus 3-4 mmol each of the remaining essential amino acids (lysine, threonine, tryptophan, and histidine). Rapid amination of the infused ketoacids occurred, as indicated by significant increases in plasma concentrations of valine, leucine, isoleucine, alloisoleucine, phenylalanine, and methionine. Glutamine, glycine, serine, glutamate, and taurine fell significantly. Blood glucose, ketone bodies, plasma free fatty acids, and serum immunoreactive insulin concentrations were unaltered. Urine urea nitrogen fell from 1.46 to 0.89 g/day on the last day of infusions; 5 days later it was still lower (0.63 g/day) and in two subjects studied for 9 and 17 days postinfusion it remained below preinfusion control values. Urine ammonia, creatinine, and uric acid were unaltered. Nitrogen balance became less negative during and after infusions. The results indicate that this mixture of essential amino acids and their keto-analogues facilitates nitrogen sparing during prolonged starvation, in part by conversion of the ketoacids to amino acids and in part by altering mechanisms of nitrogen conservation. The latter effect persists after the ketoacids are metabolized.

The effect of keto-analogues of essential amino acids in severe chronic uremia.

Alpha keto-analogues of valine, leucine, isoleucine, methionine, phenylalanine, and (in one instance) tryptophan and histidine, along with the remaining essential amino acids, were administered orally to 10 patients with severe chronic uremia fed a diet low in protein but adequate in calories. Ketoacid dosage varied from 6 to 14 g daily, as sodium or calcium salts. Net nitrogen intake, calculated as intake minus urinary protein nitrogen, averaged 1.8 g/day. The urea space was either estimated or measured with [(14)C]urea and daily changes in the body urea pool were calculated. Urea appearance was measured as the sum of urea excretion and the change in urea pool. If these ketoacids were converted to amino acids and utilized for protein synthesis, a fall in urea nitrogen appearance should occur. In five subjects, ketoacids were given for 15-18 days and then withdrawn. Urea nitrogen appearance increased 1.55 g/day on withdrawing ketoacids, and corrected nitrogen balance decreased by 1.73 g/day. In two other subjects ketoacid administration was followed, on two occasions each, by a period of administration of nine essential amino acids. In three of these four instances, urea appearance rose significantly with amino acids. In four patients studied at high blood urea levels, ketoacid treatment was relatively ineffective; two of these patients responded more favorably when studied again after peritoneal dialysis. One of these improved enough clinically to be managed as an out-patient for short intervals, despite virtual anuria. No accumulation of ketoacids in plasma or urine could be detected, and no toxicity was identified.

Synthesis of essential amino acids from their alpha-keto analogues by perfused rat liver and muscle.

Most essential amino acids can be replaced by their alpha-keto-analogues in the diet. These ketoacids have therefore been proposed as substitutes for dietary protein. In order to determine their fate in tissues of normal animals, isolated rat liver and hindquarter (muscle) preparations were perfused with keto-analogues of valine, leucine, isoleucine, methionine, or phenylalanine. When perfused at 1.5-2.0 mM, all five compounds were utilized rapidly by the liver of 48-h starved rats, at rates varying from 49 to 155 mumol/h per 200g rat. The corresponding amino acids appeared in the medium in significantly increased concentrations. Perfusion with phenylpyruvate also led to the appearance of tyrosine. Urea release was unaltered. Measurement of metabolite concentrations in freeze-clamped liver revealed two abnormalities, particularly at ketoacid concentrations of 5 mM or above: a large increase in alpha-ketoglutarate, and a moderate to marked decrease in tissue glutamine. This decrease was quantitatively sufficient to account for nitrogen appearing in newly synthesized amino acids. Isolated hindquarters of fed rats were perfused with the same ketoacids at concentrations of 1.3-8.0 mM. All were utilized at rates varying from 1.4 to 7.0 mumol/h per g muscle perfused. The corresponding amino acids were released at greatly increased rates. Alanine and glutamate levels fell in some perfusions, but the principal nitrogen donor in muscle was not identified; the content of glutamine in tissue, and its rate of release into the perfusate remained constant.

Gyrate atrophy of the choroid and retina: amino acid metabolism and correction of hyperornithinemia with an arginine-deficient diet.

Four patients with gyrate atrophy of the choroid and retina were studied, all of whom exhibited the hyperornithinemia characteristic of this disorder. Elevated plasma histidine and diminished plasma lysine and branched-chain amino acids were also noted. The renal clearances of these four amino acids were not sufficiently elevated to explain their low plasma levels. In one subject, an arginine-deficient diet led to progressive reduction in plasma ornithine from 13 times normal to the upper limits of normal, along with the disappearance of ornithinuria and lysinuria. Orally administered alpha-aminoisobutyric acid facilitated the fall in plasma ornithine by increasing renal losses of ornithine. It also increased the clearances of most other amino acids. When plasma ornithine approached normal (less than 200 microM), plasma lysine became normal, plasma arginine became subnormal, and renal clearances of basic amino acids decreased. Long-term (1.5 yr) maintenance with a diet containing 10-20 g of protein plus essential amino acids served to keep plasma ornithine at between 55-355 microM; chorioretinal degeneration did not progress and vision apparently improved.

ATP splitting and calcium binding by brain microsomes measured with a rapid perfusion method.

Rat brain microsomes, immobilized on a filter, were perfused with ATP-containing solutions in a device which made possible rapid change of perfusion media and frequent sampling of effluent. Inorganic phosphate production could be measured 10 times per sec. When ATP, sodium, or potassium was absent from the first perfusion medium and present in a second, and introduced without interrupting flow, phosphate output rose within a few tenths of a second. Inhibition by ouabain began within 0.3 sec but did not become maximal for at least 10 sec. Rapid binding of ouabain was minimal or absent, as was rapid release of ouabain on introducing potassium abruptly. Although the preparation bound some calcium reversibly, no measurable uptake of calcium occurred coincident with activation by ATP or by potassium, and no measurable release of calcium occurred coincident with the onset of ouabain inhibition. However, activation by sodium was consistently associated with simultaneous release (within < 1 sec) of calcium, averaging 46 pmole per mg of protein. Calcium release in response to sodium also occurred in the absence of ATP or in the presence of ouabain. At 0 degrees C sodium produced neither activation nor calcium release. The results are consistent with the possibility that sodium and calcium are competitively bound, even in the absence of ATP, to an active site on the enzyme distinct from the sites of potassium activation or glycoside inhibition.

Comparison of the fates of ingested leucine and ingested 2-ketoisocaproate in rats.

We previously reported that the ratio, R, of 14C to 3H in the leucine of whole body protein, measured 6 h after ingestion of [3H]leucine and [1-14C]2-ketoisocaproate is equal to ratio of the dose of leucine to the dose of 2-ketoisocaproate (KIC) (on a leucine-free diet) required to achieve the same rate of growth. To determine whether R is dependent on the interval between injection and sampling, R was measured at intervals in purified whole body protein after oral injection of these isotopes in groups of rats; it was constant from 1 h onward for 1 wk, averaging 0.64 +/- 0.01 (means +/- SEM). Thus, the extent of incorporation into the leucine of whole body protein of ingested KIC remains close to 64% of the incorporation of ingested leucine administered as such simultaneously, from 1 h onward for at least 1 wk.

Growth of rats fed by a continuous intragastric infusion containing amino acids and keto acids.

A nutrient mixture was designed to minimize electrolyte and nitrogen excretion in rats while permitting growth. It contained lysine, threonine, histidine, tryptophan, ornithine, N-free analogues of the other essential amino acids, sucrose, corn oil, minerals, and vitamins. Intragastric infusion of this mixture for 25 days into 150-g rats with no access to food or water produced an average weight gain (after a 4-day lag period) of 3.48 +/- 0.09 g/day with proportionate increase in tail length. Urinary excretion rates of N, urea, Na, K, and P became very low. Fecal N fell to 4 mg/day. Urine pH averaged 6.1. Plasma amino acid concentrations changed markedly. Carcass analysis showed that an average of 63% of the 144 mg/day of administered N was retained for growth. Thus, this nutrient mixture is unusually efficiently utilized and leads to minimal excretion of waste products.