TRANSLATIONAL PHYSIOLOGY
Role of blood cells in leucine kinetics across the human kidney

Giacomo Garibotto1, Rodolfo Russo1, Antonella Sofia1, Monica Vettore2, Laura Dertenois1, Cristina Robaudo1, Giacomo Deferrari1, Michela Zanetti2, and Paolo Tessari2

1 Nephrology Division, Department of Internal Medicine, University of Genoa, 16132 Genoa; and 2 Department of Metabolic Diseases, University of Padua, 35128 Padua, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To evaluate the role of blood cells in interorgan amino acid transport and in the estimates of regional protein turnover, we studied the effects of plasma vs. whole blood sampling on regional leucine kinetics in postabsorptive humans. Studies were carried out by combining the arteriovenous difference technique with the measurement of [14C]- and [15N]leucine isotope exchange across the human kidney, the splanchnic area, and the leg. In the kidney, whole blood-derived rates of leucine-carbon appearance, disappearance, and net balance (NB) were greater (by 3-15 times; P < 0.035) than those calculated in plasma. In addition, the net leucine-carbon (i.e., protein) balance across the kidney was negative in whole blood (-5.6 ± 1.3 µmol/min × 1.73 m2, P < 0.01 vs. 0) but neutral in plasma [-0.24 ± 1.33, P = not significant from 0; P < 0.01 vs. whole blood]. A net leucine transport out of renal cells was shown in blood but not in plasma. In contrast, rates of leucine-carbon appearance, disappearance, NB, and net transport, in both the splanchnic area and the leg, were similar in whole blood and plasma. These data suggest that blood cells play a key role in leucine transport out of the kidney and, consequently, in the leucine-derived estimates of renal protein degradation and NB, which is at variance with what is observed across the splanchnic organs or the leg. These data also emphasize the need for complete whole blood arteriovenous measurements to accurately estimate protein turnover across the kidney.

protein turnover; blood compartments; plasma; erythrocytes; amino acid transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ROLE PLAYED BY THE HUMAN kidney in interorgan amino acid metabolism and nitrogen conservation has been known for many years. Mass transfer studies have shown that there can be either a net uptake or a net release of selected amino acids across the kidney (1, 7, 11, 22, 33). With regard to the blood compartment(s) involved in such an exchange, both plasma and blood cells have to be taken into consideration (4, 11, 22, 33). Previous studies have suggested that renal uptake of individual amino acids from the artery takes place through plasma alone, whereas approx 50% of amino acid net release into the vein occurs via the blood cells (11, 33). Although these studies demonstrated that blood cells may substantially contribute to renal amino acid exchange, they could not identify the underlying kinetic mechanism(s), because they were on the basis of the measurement of net amino acid balance only.

We recently measured protein dynamics across the kidney, the splanchnic organs, and the leg in postabsorptive humans, by means of leucine and phenylalanine isotope infusions combined with the organ balance technique and whole blood sampling (30). To get further insight into the specific effect of plasma vs. whole blood sampling on the estimates of regional amino acid kinetics (and thus on the possible role of blood cells) in the present study we report the comparison between plasma and whole blood measurements of leucine and protein kinetics across the kidney, the splanchnic area, and the leg.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

Six male and five female subjects (aged 38 ± 5 yr, range 21-55 yr) participated in the study. They had been scheduled to undergo diagnostic venous catheterization assessment. The data regarding organ amino acid kinetics from whole blood sampling, for 10 of the 11 subjects in the present study, have already been reported elsewhere (30). An additional male subject who required renal vein catheterization was added to the present study. Briefly, all subjects were of normal weight (23) and had normal routine laboratory tests, glomerular filtration rates, acid-base balance, electrolyte measurements, and oral glucose tolerance tests. They were in good nutritional condition, and no significant (>3%) weight loss was noted in the previous 3 mo. None of the subjects had either history or clinical evidence of hepatic or gastrointestinal disease, congestive heart failure, kidney disease, diabetes mellitus, or other endocrinopathies. At least 1 mo before admission, they were placed on a diet providing 30-35 kcal · day-1 · kg of body wt-1 and 0.9-1 g of protein/kg of body wt, as assessed by dietary history and urea excretion.

Five subjects (3 men, 2 women) had arterial hypertension and the remaining six had cardiac valvular disease. Femoral, renal, and right-sided cardiac catheterization procedures were performed for diagnostic purposes (30). Renal vein catheterization assessment was performed on all the hypertensive patients as well as on two of the patients with valvular disease. Conversely, all six subjects with valvular heart disease and the three hypertensive patients underwent hepatic vein assessment. Thus kidney, splanchnic, and leg catheterization assessments was carried out in 7, 9, and 11 patients, respectively. In a subsequent follow-up, none of these subjects developed any metabolic disease. All subjects were informed about the nature, purpose, procedure, and possible risks of the study before their consent was obtained. The study was part of a larger protocol approved by the Ethical Committee of the Department of Internal Medicine, University Hospital of Genoa. The procedures were in accordance with the Helsinki Declaration.

Isotopes

L-[1-14C]Leucine (55 mCi/mmol; [14C]leucine, >95% pure at HPLC analysis) and [14C]sodium bicarbonate (100 mCi/mmol) were purchased from Amersham (Buckinghamshire, UK). The L-[15N]leucine [>99% mole percent enrichment (E)] was purchased from Tracer Technologies (Somerville, MA). All isotopes were proven to be sterile and pyrogen free before use.

Study Protocol

All subjects were studied in the postabsorptive state. A combination of the arteriovenous catheterization technique with the infusion of [14C]- and [15N]leucine tracers was employed, as previously described (30). Briefly, at 0800 a peripheral vein was cannulated with a Teflon catheter and used for isotope infusion. A preinfusion sample was collected to measure basal isotope enrichments (E). Thereafter, primed constant infusions of the isotopes were started by means of calibrated pumps. Priming doses of the leucine tracers (30 times the continuous infusion rate/min) as well as of [14C]bicarbonate (3 µCi) were administered as a bolus at time 0 before the start of the continuous infusion. After 120 min, catheters were placed into a radial artery and into the femoral vein. A Cobra 7F catheter (William Cook Europe, Bjaeverskov, Denmark) was then guided under fluoroscopic control through a femoral vein, either to the right ventricular cavity or to a renal vein. After the diagnostic procedure was completed, another catheter was inserted to allow simultaneous sampling from the renal and the hepatic veins. Two hundred ten minutes after the start of isotope infusions, three to four blood samples were drawn over 20-30 min into heparinized syringes kept on ice. Urine flow was collected via a bladder catheter. Arterial blood pressure and the electrocardiogram were continuously monitored during the study. Room temperature was constantly kept at 20-22°C.

Analytical Measurements

Blood samples were collected into heparinized tubes, which were gently shaken for 10-15 s. Then, 2-ml whole blood aliquots were immediately precipitated with 10% (wt/vol) cold perchloric acid (PCA). The remaining blood samples were kept on ice and centrifuged within 20-30 min at 600 g for 15 min at +4°C, for plasma separation. It has been shown that in vitro leucine transport is blocked for up to at least 30 min by low temperature (37). Both the plasma samples and the supernatant of the deproteinized blood were stored at -20°C until assay. The concentrations of leucine and of other amino acids in the PCA extract were measured by ion exchange chromatography using an amino acid analyzer and lithium buffers (33), after correction for the PCA dilution. A water content of 80% was used to calculate dilution (3). Whole blood leucine and KIC 14C-specific activities (SAs) were determined by HPLC (15, 16, 30). Whole blood KIC concentration was also determined by HPLC (16, 30) after correction of dilution with the PCA. The [15N]leucine E was determined by GC-MS as tert-butyldimethylsilyl derivative and electron impact ionization, by monitoring the fragments in the mass-to-charge ratio 201:200 (27). Isotope concentrations were calculated by multiplying either SA or E times substrate concentrations. Whole blood arterial and deep-venous concentration of [14C]bicarbonate was measured as described elsewhere (30, 32). Plasma leucine and KIC concentrations and 14C-SAs in plasma were determined by HPLC (16, 30). Amino acid concentrations in plasma were determined after deproteinization by ion exchange chromatography using an amino acid analyzer and lithium buffers (33). A water content of 94% was used to calculate dilution (3). Blood pH and PCO2 were measured at +37°C with an ABL505 apparatus (Radiometer, Copenhagen, Denmark). Oxygen saturation and Hb concentrations were measured with a hemooximeter (Radiometer). The hematocrit was determined by a microcapillary procedure.

In the present study, the arteriovenous [14C]bicarbonate (i.e., 14CO2) concentrations in plasma could not be directly measured. However, the estimation of organ leucine oxidation in plasma requires these data as well. Therefore, plasma bicarbonate concentrations were calculated from the corresponding whole blood values by applying a correction factor on the basis of the ratio between whole blood vs. plasma total bicarbonate (i.e., CO2) concentrations. This calculation is on the basis of the assumption that the diffusion of both labeled and unlabeled bicarbonate across the blood cell membrane is the same. To this purpose, total plasma and whole blood CO2 content (C-CO2) were calculated as described by Douglas et al. (10). On the basis of these measurements and calculations, we observed a constant ratio (1.2) between plasma and whole blood bicarbonate concentration, both in the veins draining each organ and in the artery (kidney, artery 1.2 ± 0.02, renal vein 1.2 ± 0.03; splanchnic organs, artery 1.2 ± 0.02, hepatic vein 1.2 ± 0.02; leg, artery 1.21 ± 0.02, vein 1.21 ± 0.02; P = NS between artery and veins). This ratio is identical to that found in the literature in peripheral venous blood (10). To further confirm these data, we determined experimentally, in the separate setting of an in vitro test, the extent and the time pattern of the equilibration between whole blood and plasma of [14C]sodium bicarbonate added to EDTA-treated venous blood samples and incubated at 37°C from 30 s to 5 min. This time interval was chosen to mimic the average delay between the actual blood withdrawal from the various catheters during the study and blood processing for 14CO2 determination (32). At various time points within this interval, samples (in duplicates) of either whole blood or plasma were taken and rapidly centrifuged. The 14CO2 content was determined in a closed system, as described elsewhere (30). We did not find any time-related change in the ratio between plasma and whole blood 14CO2 concentrations beyond the 1-min sample (data not reported). The average ratio observed in all measurements between the 1- and 5-min samples of these in vitro tests using the radioactive bicarbonate was similar to that calculated in the total CO2 (i.e., 1.20 ± 0.16, mean ± SD). Therefore, we calculated 14CO2 plasma concentrations in both the arterial and the venous samples across each organ by multiplying the whole blood data times 1.2. Expired 14CO2 was collected as reported elsewhere (30).

Plasma and Blood Flow Measurement

Renal plasma flow was measured by the clearance of sodium PAH (28, 33). Three sequential clearance periods of 20 min each were obtained. Renal plasma flow was calculated from the clearance and the extraction of PAH by using Wolf's equation (38). Renal whole blood flow was calculated by dividing plasma flow by (1 - hematocrit). Hepatic plasma flow was measured by a primed continuous intravenous infusion of indocyanine green (5). Four simultaneous arterial and hepatic venous plasma samples were collected at 5- to 10-min intervals. After centrifugation, absorbance in the plasma at 805 nm was determined (5). Leg blood flow was estimated by the arteriovenous difference of oxygen concentration and VO2 (13). Leg and splanchnic blood flows were calculated by multiplying whole blood flow times (1 - hematocrit).

Calculations and Data Presentation

All the kinetic calculations on the basis of both whole blood and plasma data were performed under steady-state conditions (31, 32). Interorgan fluxes were determined by using plasma flow, concentrations, and SAs or enrichments in plasma (for the calculations in plasma) or whole blood flows, concentrations, and SAs or enrichments in whole blood (for calculations in whole blood), respectively.

Organ leucine kinetics, i.e., the rates of leucine-carbon appearance (Ra), disappearance (Rd), oxidation, disposal into protein synthesis (PS), and net balance (NB), were first calculated by using a noncompartmental arteriovenous approach, as described elsewhere (30). In the calculations, we also included the leucine deamination product alpha -ketoisocaproate (KIC; both unlabeled and labeled) to account for the overall leucine-carbon (i.e., leucine+KIC) organ kinetics. Indeed, KIC kinetics are different across different organs, as reported previously (30), and they may thus significantly affect the estimate of organ total leucine carbon Ra and Rd. Organ leucine oxidation was calculated by dividing the measured (for whole blood) or the calculated (for plasma) arterio-venous [14C]bicarbonate concentrations, by whole blood or plasma venous [14C]KIC-SA, respectively, and then by multiplying this ratio times flow. The rate of nonoxidative leucine disposal into PS was then calculated by subtracting oxidation from Rd. Leucine NB was calculated from the difference between PS and Ra.

Second, we also analyzed our data with a six-compartmental model of organ leucine-carbon kinetics (31). This model is on the basis of the simultaneous infusion of [14C]- and [15N]leucine tracers (31). From the differential behavior between these tracers, it is possible to also estimate the rates of leucine inflow and outflow between the extracellular and the intracellular space(s), as well as the rates of intracellular leucine and KIC interconversions, leucine release from proteolysis, oxidation, and disposal into PS (31). The use as the precursor pool of [14C]KIC-SA in the vein draining each organ allows a reasonable estimate of the intracellular leucine kinetics (25, 30-32, 35, 36). The assumptions and the equations of this model have been reported elsewhere (30, 32). We report here only the explanations of the model abbreviations used. Thus F1 and F4 indicate arterial leucine delivery to and venous leucine release from the sampled organ, respectively. F2 and F3 indicate leucine inflow and outflow across the cell membranes, respectively. F5 and F6 indicate leucine release from organ proteolysis and disposal into PS, respectively. F7 and F8 indicate leucine deamination to KIC and KIC reamination to leucine, respectively. F9 indicates leucine oxidation. F10 and F11 indicate arterial KIC delivery to and venous KIC release from the sampled organ, respectively. Finally, F12 indicates the leucine directly transferred from the artery to the vein without being metabolized.

All kinetic data are expressed as micromoles per minute times 1.73 m2 of body surface, i.e., they were normalized over this estimate of body size to also account for differences in organ size. Data are expressed as means ± SE.

The statistical analysis was performed by using two-way ANOVA (Statistica, version 4, StatSoft, Tulsa, OK), for repeated measurements, to analyze simultaneously two pairs of related sets of data [i.e., 2 sets of arterial measurements in whole blood and plasma and 2 sets in the vein; organ protein degradation and synthesis measured from plasma and whole blood data, and similarly, organ leucine delivery (F1) and release (F4), leucine deamination (F7) and reamination (F8), and transmembrane leucine inflow (F2) and outflow (F3)] (31). In addition, where not otherwise stated, paired t-tests (Student's t-test when data distribution was normal or Wilcoxon test when it was not normal) were employed to compare individually two sets of paired data. A P value <0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Substrate and Isotope Concentrations, SAs, and Enrichments in Blood and Plasma

Arterial concentrations of both leucine and KIC were higher in plasma than in whole blood (Table 1). Also arterial leucine and KIC isotope concentrations were greater in plasma than in whole blood. In contrast, there were no differences either in the [14C]leucine and [14C]KIC-SAs or in the [15N]leucine E between arterial blood and plasma samples.

                              
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Table 1.   Substrate concentrations, isotope concentrations, specific activities, and [15N]leucine mole percent enrichment in whole blood and plasma

Kidney. There was a significant effect of blood vs. plasma sampling on the arteriovenous differences of leucine concentrations (P < 0.015 by ANOVA; Table 1). The whole blood leucine concentrations in the renal vein were slightly, although significantly, greater (P < 0.05 by paired t-test) than those in the artery, whereas they were similar in plasma (P = NS). Both arterial and venous plasma concentrations were greater than those in blood (P < 0.05). These findings indicated a net leucine release from the kidney only through the whole blood compartment. Opposite to leucine, the renal vein KIC concentrations were lower than in the artery in both whole blood and plasma samples (P < 0.0001 by ANOVA; Table 1), suggesting a net renal KIC uptake from both compartments. The whole blood [14C]- and [15N]leucine and [14C]KIC isotope concentrations, both arterial and venous, were greater (P < 0.05 or less by ANOVA) than those in plasma. As concerns the arteriovenous differences in isotope concentrations, the [14C]- and [15N]leucine and the [14C]KIC concentrations in the renal vein were lower (P < 0.05 or less by ANOVA) than those in the artery in both whole blood and plasma, indicating the renal leucine and KIC extraction through both compartments. The [14C]leucine-SA and [15N]leucine E in the artery were greater than those in the renal vein in either whole blood or plasma (P < 0.003 by ANOVA), indicating a simultaneous leucine release by the kidney. There was a significant effect (P < 0.015 by ANOVA) of blood vs. plasma sampling on the arteriovenous differences of [14C]KIC-SA. The arterial [14C]KIC-SA was greater than the venous one in whole blood (P < 0.05), whereas the arterial values were lower than the venous ones in plasma (P < 0.05). Both whole blood and plasma [14C]bicarbonate concentrations in the renal vein were significantly greater than in the artery, indicating a net renal production of [14C]bicarbonate. The [14C]bicarbonate concentrations calculated in plasma were 20% greater than those measured in whole blood, on the basis of the assumptions (see METHODS). Blood and plasma flow across the kidney were 963 ± 71 and 621 ± 44 ml/min × 1.73 m2, respectively.

Splanchnic organs. Leucine concentration in the hepatic vein was lower than in the artery (P < 0.001 by ANOVA; Table 1) in both whole blood and plasma, indicating net amino acid uptake from both compartments. Leucine concentrations in plasma were greater than in whole blood (P < 0.03 by ANOVA) in both artery and vein. In contrast, KIC concentrations were higher in the hepatic vein than in the artery in either whole blood or plasma (P < 0.003 by ANOVA), indicating a net KIC release from both compartments. The [14C]- and [15N]leucine isotope concentrations in the artery were significantly greater (by 20-25%, P < 0.001 by ANOVA) than in the hepatic vein in both whole blood and plasma, consistent with leucine uptake. The venous [15N]leucine isotope concentrations in plasma were greater (P < 0.025 by t-test) than those in blood, whereas these differences did not attain statistical significance in the artery. In contrast, the [14C]KIC isotope concentrations in the hepatic vein were significantly greater (P < 0.01 by ANOVA) than in the artery in both whole blood and plasma, suggesting a KIC production from leucine within the splanchnic area, as well as through both the blood and the plasma compartments. The [14C]leucine-SA and the [15N]leucine E in the artery were greater than in the hepatic vein in both plasma and whole blood (P < 0.001 by ANOVA), without differences between the two compartments, indicating a simultaneous substrate release. In contrast, there was no difference in the [14C]KIC-SA between artery and hepatic vein in either whole blood or plasma samples. The [14C]bicarbonate concentrations in the artery were greater than in the hepatic vein in both whole blood and plasma. Blood and plasma flows across the splanchnic organs were 1,307 ± 107 and 834 ± 44 ml/min × 1.73 m2, respectively.

Legs. The leucine concentrations in the femoral vein were slightly, although significantly, greater than in the artery (Table 1) both in plasma and whole blood (P < 0.001 by ANOVA), indicating a net amino acid output. The whole blood leucine concentrations in both artery and vein were greater than those in plasma (P < 0.05 by ANOVA). The concentrations of KIC in the artery and in the femoral vein were similar in both plasma and whole blood, indicating no net KIC production by the leg. The [14C]- and [15N]leucine isotope concentrations were approx 25% greater (P < 0.001 by ANOVA) in the arterial than in the venous blood both in plasma and whole blood compartments, indicating amino acid uptake. The [14C]KIC isotope concentrations were, however, not different between artery and vein in either whole blood or plasma, suggesting no KIC utilization from extracellular sources by the leg. The [14C]leucine-SA and the [15N]leucine E in the femoral vein were significantly lower (P < 0.001 by ANOVA) than those in the artery both in plasma and whole blood, indicating simultaneous dilution of the tracers by the release of unlabeled leucine. There was no difference in the [14C]KIC-SA between whole blood and plasma in either the artery or the vein. Whole blood and plasma [14C]bicarbonate concentrations in the femoral vein were 10% greater than in the artery both in whole blood and in plasma, consistent with a net production of 14CO2 from the oxidation of [14C]leucine. Blood and plasma flows across the leg were 927 ± 92 and 583 ± 60 ml/min × 1.73 m2, respectively.

Leucine Kinetics Across the Organs with the Noncompartmental Model

Kidney. Leucine-carbon Ra (13.5 ± 3.8 µmol/min × 1.73 m2) and Rd (13.9 ± 3.9) were greater (P < 0.035 by ANOVA) when calculated in whole blood than in plasma (Ra = 1.4 ± 1.8 and Rd = 5.7 ± 1.9 µmol/min × 1.73 m2, respectively) (Fig. 1). Leucine oxidation was also greater (P < 0.02) when measured in blood than in plasma (7.0 ± 0.8 and 4.6 ± 0.6 µmol/min × 1.73 m2, respectively). The rate of PS estimated in whole blood tended to be greater than that estimated in plasma (6.2 ± 4.3 and 1.7 ± 1.9 µmol/min × 1.73 m2, respectively), although not significantly. As a result, however, the difference between Ra and PS, indicating the NB of leucine-carbon into/out of renal proteins, was negative in whole blood (-5.6 ± 1.2 µmol/min × 1.73 m2, P < 0.01 vs. 0), whereas it was not different from 0 in plasma (-0.24 ± 1.33, P < 0.01 vs. whole blood).


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Fig. 1.   Leucine-carbon rate of appearance (Ra), disappearance (Rd), oxidation (Ox), disposal into protein synthesis (PS), and net balance (NB) across the kidney, calculated with a noncompartmental model including both leucine and alpha -ketoisocaproate acid kinetics, using either whole blood data (left) or plasma data (right). **P < 0.02, *P < 0.05 whole blood vs. plasma.

Splanchnic organs. There was no difference between leucine-carbon Ra or Rd in either whole blood (30.4 ± 6.6 and 34.4 ± 7.4 µmol/min × 1.73 m2, respectively) or plasma (27.2 ± 4.3 and 30.4 ± 6.5 µmol/min × 1.73 m2, respectively) nor between the whole blood and the plasma estimates of Ra and Rd. Leucine oxidation was slightly, although significantly, greater (P < 0.05) in blood than in plasma (3.9 ± 1.8 and 3.4 ± 1.6 µmol/min × 1.73 m2, respectively). As a result, neither PS nor NB was different between blood (PS = 30.1 ± 7.8 and NB = -0.74 ± 3.3 µmol/min × 1.73 m2) and plasma (PS = 29.7 ± 5.4 and NB = 0.91 ± 2.1 µmol/min × 1.73 m2). Notably, NB was not different from 0 in either plasma or whole blood.

Legs. Leucine-carbon Ra was greater (P < 0.02 by ANOVA) than Rd in both blood (Ra = 23.3 ± 4.4 and Rd = 20.1 ± 4.3 µmol/min × 1.73 m2) and plasma (Ra = 17.7 ± 1.6 and Rd = 15.1 ± 1.1 µmol/min × 1.73 m2). However, no differences were detected in both Ra and Rd between blood and plasma measurements. Leucine oxidation was greater (P < 0.02) in blood (4.9 ± 0.1) than in plasma (3.7 ± 0.7). As a result, neither PS nor NB was different between whole blood (PS = 15.7 ± 4.9 and NB = -7.7 ± 2.3 µmol/min × 1.73 m2) and plasma measurements (PS = 12.5 ± 0.8 and NB = -6.5 ± 1.5 µmol/min × 1.73 m2).

Leucine Kinetics Across Organs with the Compartmental Model

Kidney. There was a significant effect (P < 0.015 by ANOVA) of whole blood vs. plasma sampling on the rates of leucine disposal into PS (the F6 model parameter) and of proteolysis (F5), which were both several times greater in blood than in plasma (Table 2). F6 was lower (P < 0.015 by paired t-test) than F5 in whole blood, whereas these rates were not different in plasma. The resulting NB [i.e., (F6 - F5)] was neutral in plasma but negative in whole blood (P < 0.025, plasma vs. whole blood), thus indicating a net renal protein loss occurring in blood but not in plasma. Similarly, there was a significant effect (P < 0.01 or less by ANOVA) of whole blood vs. plasma sampling on the rates of arterial leucine delivery to (F1) and release from (F4) the kidney, of leucine inflow (F2) and outflow (F3) between the extracellular and the intracellular space(s), of leucine bypassing intracellular metabolism (F12), and of leucine oxidation (F9). All these rates were greater when using the whole blood data than when using the plasma data. The difference between F2 and F3 (indicating net leucine intracellular retention) was negative (i.e., indicating net release) in whole blood but not different from 0 in plasma (Table 2). In contrast, the leucine deamination and reamination rates, as well as net leucine deamination [i.e., (F7 - F8)], were not different between whole blood and plasma (Table 2).

                              
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Table 2.   Leucine and KIC kinetics across kidney, splanchnic area, and leg, measured in whole blood and in plasma

Splanchnic bed. There was no difference between PS (F6) and degradation (F5) across the splanchnic area, estimated either in whole blood or plasma. The resulting net leucine balances were not different from 0 in both compartments (Table 2). No effect of blood vs. plasma sampling was also found in the rates of leucine delivery (F1) and release (F4); F4 was lower than F1 in both compartments (P < 0.0015 by ANOVA). The rate of leucine inflow (F2) across the cell membrane was greater than outflow (F3) in both whole blood and plasma, either rate being greater (P < 0.05 by ANOVA) in blood. The net leucine retention [i.e., (F2 - F3)] was similar in blood and plasma. The rates of leucine deamination (F7) and reamination (F8) were greater in blood than in plasma (P < 0.02 by ANOVA); F7 was greater than F8 in both compartments (P < 0.001 by ANOVA). Net deamination [i.e., (F7 - F8)], as well as leucine oxidation, were significantly greater (P < 0.02 or less ) in whole blood than in plasma.

Legs. Protein degradation (F5) was greater than synthesis (F6) in both blood and plasma (P < 0.0001 by ANOVA) (Table 2), although no effect of blood vs. plasma sampling was found (P = NS by ANOVA). The resulting net leucine balance was negative, and to a similar extent, in both compartments. No effect of blood vs. plasma sampling was also found in the estimates of F1-F4, F7, and F8. F12 was greater in blood than in plasma. However, F4 was >F1, and F3 was >F2 (P < 0.025 by t-test), only in plasma, whereas F7 was >F8 to similar magnitudes in both compartments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Elwyn (11) suggested that red cells act as carriers of amino acids from one site in the body to another. In previous reports on the basis of measurements of organ net amino acid balance in both whole blood and plasma (11, 12, 33), it was observed that blood cells and plasma play an independent, and sometimes opposing, role in interorgan amino acid exchange. However, no information on the mechanisms of these effects or on regional amino acid kinetics was obtained in those studies because no tracer was infused.

The main findings of the present study are that the type of arteriovenous sampling (i.e., whole blood vs. plasma) markedly affects leucine kinetics and net protein balance specifically across the kidney. Indeed, the rate of renal protein degradation and, to a lesser extent, that of PS were greater in blood than in plasma. As a consequence, the renal net protein balance was negative in blood but neutral in plasma. The higher rates that were measured in blood compared with plasma were not due to the higher whole blood than plasma flow, because no analogous differences were detected across either the splanchnic area or the leg. Therefore, these data suggest that blood cells (which are included in the whole blood but not in plasma analyses) play a specific role in renal leucine release and/or transport out of this organ. This conclusion is also supported by the leucine arteriovenous concentration differences, which were negative in whole blood but neutral in plasma.

Leucine is an essential amino acid, and in the postabsorptive state, its only sources are body protein stores. After [l4C]leucine tracer infusion, whole blood leucine-SA in the veins of all three catheterized organs was substantially lower than in the artery because of isotope dilution by unlabeled leucine, which derives from tissue protein degradation (31). Because there is no proteolysis in mature erythrocytes (18), and red cells represent the bulk of blood cells, it is reasonable to assume that the leucine-SA within the blood cells is not affected by intraerythrocyte structural protein degradation. In support of this assumption, it was shown that leucine fully equilibrates between plasma and erythrocytes over the course of systemic leucine tracer administration and sampling (8).

With regard to the possible mechanisms for the preferential leucine transport outside the kidney by means of blood cells, the use of a compartmental model may provide some information. There was a significant effect of whole blood vs. plasma sampling on the rate of leucine transmembrane exchange across renal cells. Indeed, F3 (i.e., leucine outflow) was greater than inflow (F2) in whole blood, whereas they were similar in plasma, thus resulting in a net leucine outward transport from renal cell(s) only through the blood compartment (Table 2). Therefore, blood cells appear to act as carriers of leucine out of the kidney. Such behavior may have significantly affected the estimates of the rates of protein degradation and NB calculated from whole blood measurements, specifically in this organ. In contrast, analogous differences in leucine transmembrane exchange were not observed across the splanchnic area or the leg (Table 2).

What are the process(es) that are unique to the kidney that might generate the observed preferential transport of leucine out of the renal tissue to venous blood cells? As mentioned above, the kidney plays a major role in the handling of peptides and/or of low- molecular-weight proteins, which are filtered and subsequently reabsorbed (17, 32, 33). The kidney, unlike other organs, is able to accumulate dipeptides against a concentration gradient (9, 14), and the dipeptide hydrolase activity in renal cells far exceeds the activity of this enzyme in the liver and muscle (19, 21, 22). The kidney is also characterized by one of the highest fractional protein turnover rates (30). All these processes could result in a huge load of amino acids to the renal tubule cells. Preferential transport of free amino acids out of tubular cells through the red cells might allow for more efficient amino acid recovery into the systemic circulation, thus minimizing the loss from body pool(s). Indeed, red cells are quite permeable to leucine; the major route of amino acid entry is a saturable system, which involves facilitated diffusion (34). In addition, an Na-dependent L-transport system has been observed in the red cells (34). Amino acid transport by means of the blood cells, in association with the high organ blood flow, might ensure fast and efficient delivery of the amino acid(s) into the renal vein. It is noteworthy that the human kidney has the greatest blood flow rates relative to organ mass and that the mean transit time across the kidney is only 4-6 s for plasma and 4-5 s for red cells (2, 7).

In contrast to the kidney, in the splanchnic bed and in the leg no differences between whole blood and plasma sampling were observed in the rates of protein degradation, synthesis, and NB. However, in the splanchnic area, the rate of leucine conversion to KIC (F7) and the rate of KIC conversion to leucine (F8) in blood were higher than in plasma. These observations suggest that the higher blood flow than plasma flow and/or of the red cells themselves might have an important effect on the rates of leucine transport and transamination in this specific site. In particular, the high leucine transaminase activity within splanchnic organs (mostly the liver) (17, 20) likely couples with the higher leucine delivery from blood compared with plasma and yields the high rates of leucine transamination observed in whole blood data.

The irreversible (i.e., net) disappearance of the [15N]leucine isotope across the organs is essential for an accurate estimate of transamination rates using the compartmental model reported here. The net extraction of the [15N]leucine isotope across the kidney was indeed very small (Table 1), as was the [14C]leucine isotope, at variance with larger extractions observed across the other two catheterized organs. We cannot rule out that significant [15N]leucine isotope recycling and/or limited dilution of the 15N tracer took place in the kidney because of the high renal flow relative to organ mass and/or to the small, intrarenal leucine pool (24). This would lead to lower estimates of both transmembrane leucine exchange and transamination rates, which are more marked in the blood than in the plasma compartment, possibly because of the limited time allowed for leucine to equilibrate between plasma and red cells. On the other hand, the net transport as well as net deamination rates are unaffected by possible isotope recycling, as discussed previously (31). Therefore, some of the differences we observed between whole blood and plasma measurements with regard to specific leucine kinetic parameters might be considered qualitative rather than quantitative mainly in the kidney. The possibility that our results on blood vs. plasma sampling on kidney protein turnover is due to delayed equilibration of leucine in the blood perfusing kidney tissue is unlikely, because it should have been associated with a lower blood leucine concentration in the renal vein (which was increased) and with reduced whole blood appearance of leucine in the renal vein (which was increased).

The findings of the present study have methodological implications, because plasma or mixed plasma/whole blood, rather than whole blood measurements, have been frequently used in studies on regional organ amino acid kinetics (6, 29, 39). In addition, this study provides new information on the role played by the kidney in body protein metabolism, particularly on the possible links between kidney protein metabolism and renal damage (26). Indeed, in patients with chronic renal failure, increased protein handling by renal tubules might cause significant tubulointerstitial damage (26) and functional impairment of this organ. It is noteworthy that in these patients nitrogen balance across the kidney is even more negative than it is in normal conditions (33), thus suggesting an extensive degradation of proteins by tubular cells. The results of the present study add some mechanistic insight into the above observations. They also indicate the need for further investigations of renal protein dynamics, both in renal insufficiency and during renal tubule protein overload, to shed further light on the relationships between renal protein metabolism and function in chronic renal diseases.

In conclusion, this study shows that whole blood vs. plasma sampling results in important differences in leucine release from protein degradation and NB across the kidney but not across the splanchnic area or the leg. Therefore, whole blood sampling is necessary to measure correctly renal protein kinetics using the catheterization, isotope infusion technique.


    ACKNOWLEDGEMENTS

This study was supported by grants from the Universities of Genoa and Padua and the Ministero dell' Università e della Ricerca Scientifica e Tecnologica (MIUR 2001 and FIRB).


    FOOTNOTES

Address for reprint requests and other correspondence: G. Garibotto, Nephrology Div., Dept. of Internal Medicine, Univ. of Genoa, Viale Benedetto XV No. 6, 16132 Genoa, Italy (E-Mail: gari{at}unige.it).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

August 13, 2002;10.1152/ajprenal.00230.2001

Received 24 July 2001; accepted in final form 23 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 283(6):F1430-F1437
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