Brain metabolism of amino acids and ammonia in patients with chronic renal insufficiency

Kidney International, Vol. 20 (1981), pp. 505—5 10

Brain metabolism of amino acids and ammonia in patients with chronic renal insufficiency GIAcoMo DEFERRARI, GIAcoMo GARIBOTTO, CRISTINA ROBAUDO, GIAN MARCO GHIGGERI, and ALBERTO TIzIANELL0 Istituto Scientifico di Medicina Interna, Section of Nephrology, University of Genoa, Genoa, Italy

and phosphate balances, and the presence of hypertension and uremic toxins themselves 11—3] may have important pathogenic

Brain metabolism of amino acids and ammonia in patients with chronic renal insufficiency. The cerebral metabolism of amino acids (AA) and ammonia in the postabsorptive state was evaluated in 8 subjects with normal renal function and in 6 patients with chronic renal insufficiency (CR1) by measuring the differences between the arterial and the internal

roles.

jugular venous concentrations of free AA and ammonia. In normal conditions, the brain extracts serine, glutamine, proline, glycine, Valine, and

isoleucine, leucine, and lysine. In CR1, cerebral glycine

Increased levels of 5-hydroxyindoleacetic acid and homovanillic acid in the cerebral spinal fluid (CSF) of uremic patients

[41, and high serotonine and low dopamine contents in the cerebral tissue of patients who died while in uremic coma have been reported [5]. In addition, cerebral glucose consumption is decreased in patients with chronic renal insufficiency (CR1) [6].

uptake increases, valine and isoleucine extraction decreases, glutamine uptake disappears, and ammonia extraction becomes evident. The cerebral extraction of glycine is correlated with the arterial concentration of glycine, serine, and branched-chain AA. The extraction of '/2cystine is correlated with the arterial concentration of and tyrosine. Finally, the extractions of valine and ammonia are correlated with the arterial concentration of valine and ammonia,

More recently, changes of CSF amino acid (AA) conceritrations, unrelated to alterations in their blood concentrations,

respectively. It follows that alterations of blood AA and ammonia concentrations observed in CR1 markedly affect the cerebral uptake of some AA and ammonia. The lack of cerebral glutamine extraction might be due to an enhanced production and/or, more likely, to an impaired utilization of this AA by the brain. Data reported here suggest that in CR! cerebral nitrogen metabolism is altered; such alterations may play a pathogenic role in uremic encephalopathy.

have been detected in patients with CR! [7]. The all data clearly suggest that cerebral metabolism, including AA and neurotransmitter metabolism, is altered in renal insufficiency.

The concentration of AA in the blood is one of the major factors controlling their transport into the brain [8, 9]. In CR!, the arterial blood concentration of several AA's is altered quite early and in the absence of malnutrition [10]. Accordingly, these concentration changes may affect AA's entry into the brain and, consequently, cerebral AA metabolism. It may be hypothesized that such alterations play an important role in the pathophysiology of uremic encephalopathy. Studies reported here were undertaken to investigate cerebral AA and ammonia metabolism in patients with CR!, by measuring the arteriovenous difference of these metabolites across the

Métabolisme cérébra! des acides amines et de l'ammoniaque chez les malades atteints d'insuffisance rénale chronique. Le m&abolisme céré-

bral des acides aminés (AA) et de l'ammoniaque dans Ia phase post absorption a été étudié chez 8 sujets ayant une fonction renale normale et 6 malades atteints d'insuffisance rénale chronique par Ia mesure de Ia difference de concentration arterio-vieneusejugulaire interne des AA et de l'ammoniaque. Dans les conditions normales le cerveau extrait Ia sérine, Ia glutamine, La proline, Ia glycine, Ia valine, Ia cystine, l'isoleucine, Ia leucine et Ia lysine. Dans I'insuffisance rénale chronique Ia captation cérébrale de glycine et de '/2 cystine augmente, l'extraction de valine et d'isoleucine diminue, Ia captation de glutamine disparait et l'extraction d'ammoniaque devient évidente. L'extraction cérébrale de glycine est corrélée a Ia concentration artérielle de glycine, de sérine et des AA a chaine latérale. L'extraction de Ia cystine est corrélée a Ia concentration artérielle de cystine et de tyrosine. Enfin les extractions de valine et d'ammoniaque sont corrélées respectivement avec les

brain.

Methods

Patients. Two groups of patients were studied. The first

concentrations de valine et d'ammoniaque. II en résulte que les modifications des concentrations sanguines des AA et de l'ammoniaque observees dans l'insuffisance rénale chronique affectent de facon im-

portante Ia captation cérébrale de certains AA et de l'ammoniaque. L'absence d'extraction cérébrale de glutamine pourrait être due a une augmentation de Ia production et/ou, plus probablement, a une utilisa-

tion defectueuse de cet acide amine par le cerveau. Les résultats

group consisted of eight patients, aged 29 toSS yr, with cardiac valvular diseases. Routine laboratory tests, acid-base and electrolyte measurements, and renal function tests were normal. In these patients, a right-sided cardiac catheterization was considered necessary for diagnostic hemodynamic evaluation. The second group consisted of six patients, aged 22 to 56 yr, with chronic glomerulonephritis and renal insufficiency. Azotemia had been present from 3 to 36 months. GFR was 18.9 3.5

rapportés suggerent qu'au cours de l'insuffisance rénale chronique Ic métabolisme cérébral de l'azote est modiflé. Ces modifications pourraient jouer un role pathogenique dans l'encéphalopalhie urémique.

Received for publication October 27, 1980 and in revised form February 24, 198!

The pathophysiology of uremic encephalopathy is still poorly understood. Alterations of hydric volumes, of sodium, calcium 505

0085-2538/81/0020-0505 $01.20

© 1981 by the International Society of Nephrology

Deferrari ci a!

506

Table 1. Arterial concentrations and arterial-jugular venous differences of free amino acids and ammonia in 8

subjects with

normal renal

function and in 6 patients with chronic renal insufficiency (CR1) A-V differences, p.mo!eslliter

Arterial concentrations, p.rnoles/liier

In controls Taurine Aspartate Threonine

99.3

4.75

59.3 138.7

7.57

495.7 212.6

26.49

24.06

NS <0.05 NS <0.05 NS NS NS <0.01

300.9 272.0 + 61.4 154.4 83.8 47.6 86.4 32.2

17.02

<0.01

24.78 5.30 12.69 7.23 6.48

<0.1 <0.001 <0.05 <0.001 NS NS <0.05 NS NS NS <0.025 NS <0.025

18.57

216.6

30,78

80.6

10.06

Asparagine Glutamate° Glutamine'

118.4 + 11.43 108.2 7.67 5.92 117.6 4.93 53.0 148.1 9.66 530.0 19.55

Proline

131.1

12.68

Glycine Alanine Citrulline Valine Cystine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Arginine Ammonia

263.0 210.0 32.1 182.0 45.9 49.9 97.7 41.8 41.2 73.2 142.7 66.8 62.0 43.7

13.06 15.14

Serine

207.2

2.47

4.89 4.65 1.67 2.89 2.18 1.86

4.65 8.00 2.46

3.80 4.01

In controls

In CR1

107.1 -'- 9.69

9.84

10.09 3.75

49.1 - 6.30 70.8

4.34

144.5

10.18

85.0 6.46 66.9 + 5.38 69.5 7.64

+ 7.9 + 2.1 + 2.0 + 3.7 + 0.9 + 2.3

4.73 1.16

+ 13.4

4.46

1,05 1.14° 0.71 1.09

+ 6.1 ÷ 1,66k + 5.2 l.09'° — 0.1 + 1.49

+ 1.6 0.72 + 9.0 1.69' + 2.9 0.33' + 3.1 0.60' + 6.1 0.70' + 0.7 0.56 + 1.3 + 0.60 + 1.1 0.52 + 3.2 0.96 + 1.3 0.57 + 1.9 1.01 — 1.0 + 1.16

In CR1

+ 0.6 + 2.1 + 3.9 + 3.4

6.14 2.44

— 0.1

+ 5.6

0.99 2.47 4.05 1.46'

NS <0.05 NS

+ 11.7

2.79w

<0.05

+ 0.6

3.63 0.74

NS NS <0.05 <0.025 <0.1 NS NS NS NS NS NS NS <0.05

— 0.5 — 1.0

1.5

+ 4.0 + 6.2 + 1.5 + 4.8 — 0.1

+ 1.2 + 3.0 + 3.6 + 0.4

1.81

l.24

1.26 1.53" 0.581

0.57' 0.38 0.59 1.40 1.59t 1.41

— 1.3

1.41

+ 9.0

3.80°

NS NS NS

NS NS

Values are given as the means SEM. Probability that patients with CR1 do not differ from controls Enzymatically determined. d Determined only in 7 subjects. Determined in 2 additional patients Probability that A-V difference does not differ from zero: P < 0.05; P <0.025; h P < 0.01; P < 0.005; P < 0.001. b

1.73 m2; blood urea was 21.2 2.4 mmoles/liter; arterial bicarbonate was 17.4 0.9 mmoles/liter; and hematocrit was 32.8 1.7%. Serum sodium and potassium, and serum albumin were in the normal range. Three patients showed bone alterations that were more severe than could be expected on the basis of the degree and the duration of their renal insufficiency and that were attributed to hyperparathyroidism. Innominate, internal jugular, and thyroid vein catheterization for parathormone measurements was carried out to localize the hyperfunctioning parathyroid tissue, in view of a possible parathyroidectomy. The other three patients had cardiac valvular diseases, and a right-sided cardiac catheterization was considered necessary for diagnostic hemodynamic evaluation. Neither group of patients had history or evidence of neurologic disorders, congestive heart failure, hepatic or pulmonary diseases, or diabetes mellitus. All patients were on a diet that provided 35 to 40 kcal and 0.8 to 0.9 g of protein per kilogram of body wt per day. They were in good nutritional balance and were actively employed at the time of the study. All patients were informed of the nature, purpose, procedure, and possible risks before their voluntary consent was obtained. Procedure. All patients were studied in the postabsorptive mi/mm

•

state. A teflon catheter was inserted percutaneously into a peripheral artery. A Cournand no. 6 or 7 F catheter (USCI International, Murray Hill, New Jersey) was then guided under fluoroscopic control through an anteeubital vein to the superior bulb of an internal jugular vein or to the right ventricular cavity. During the removal of the catheter from the ventricular cavity, it was inserted into the superior bulb of an internal jugular vein. The position of the catheter was ascertained visually with image

intensifIcation before each blood withdrawal. The catheters were kept patent by flushing with saline intermittently. From each subject, two or three sets of blood samples were obtained simultaneously from a peripheral artery and an internal jugular vein for the measurement of arterial-jugular venous (A-V) differences of AA and ammonia. Blood was withdrawn by heparinized syringes kept in ice. Arterial blood pressure and electrocardiogram were continuously monitored during the study. Analysis. The methods used for preparation of samples and determination of whole blood AA's are reported elsewhere [11].

Lithium buffers instead of sodium buffers were used for AA analyses in this study. Accordingly, treatment of the whole blood supernate with sodium suiphite was omitted. Aspartate was measured on supernate buffered up to a pH of 7 immediately after the deproteination, and stored at —25° C. The assay was

performed at least 20 days after the withdrawal to avoid the overlapping of the glutathione peak with the aspartate peak. Glutamine and glutamate were measured enzymatically, as reported elsewhere [11].

Ammonia was determined on whole blood. Samples were deproteinized at +4° C with 0.3 M sodium tungstate and 0.5 M sulphuric acid immediately after the withdrawal. The proteinfree supernate was stored at —25° C and assayed according to Chaney and Marbach [12] within 12 hours. An eightfold concentration of phenol and hypochlorite reagents was used. Blanks, samples, and standards were all in triplicate. Sodium thiosuiphate method was used for GFR measurement according to Brun [13]. Urea was determined enzymatically [12]. Arterial blood pH and Pco2 were estimated at 37° C with

507

Brain amino acid and ammonia metabolism in renal failure y = —10.397 + 27.933x )r = 0.780; P

PHM-72 BMS-3 apparatus (Radiometer Co., Copenhagen, Den•

mark). Arterial bicarbonate was calculated using the Henderson-Hasselbalch equation. Hematocrit was determined by a microcapillary procedure.

The nitrogen balance across the brain was calculated by

adding the nitrogen contributed by individual AA and ammonia which were significantly extracted by the brain. Controls and patients with CR1 were compared by analysis of variance by using a completely randomized design; a randomized block design was applied to the analysis of variance for the paired data [14]. Analysis of simple regression and correlation [14] was used to evaluate the dependence of A-V differences of

+25

assumed to be in competition for the same transport system across the blood-brain barrier (BBB) [15]; and (c) the ratio of its

<0005)

0.089x )r 0.664; P <0.025)

(A)

(B)

8= 0

0

. i+15

. +5

S•

—5

200

each AA on: (a) its arterial concentration; (b) the ratio of its concentration to the concentration of each individual AA that is

—16.690 +

y

300 Glycine, pmo/es/Iiter

400

0.4

0.8

1.2

Gycine/serino + BCAA

1. Relationships between glvcine A-V di:ffrence and A arterial glycine concentration and B the ratio of arterial glycine to the sum of Fig.

arterial concentrations of serine and branched-chain amino acids arterial blood concentration to the sum of the concentration of (BCAA) in 7 subjects with nor/na! rena/function (solid circles) and in 6

all AA that are assumed to be in competition for transport. patients with chronic renal insitfficiencv (open circles). Analysis of multiple regression [14] was used to estimate the dependence of A-V differences of each AA on the arterial AA

concentrations that show a simple correlation with the A-V

y = 0.2158 eOl9b0 )r = 0.660; P <0.02)

differences, as obtained from analyses carried out according to a and b. The relationship between A-V differences of ammonia and its arterial blood concentration was studied by analysis of 0'a

simple regression and correlation. Analyses of simple and =E 0a multiple regression were carried out by considering subjects with normal renal function and patients with CR1 as a single >.s group. Values are given as the means SEM. The absolute rates of metabolite exchanges across the brain could not be calculated because blood flow was not measured.

>

+20

+15

S

.

+10

+5

I it

Results I In normal conditions, the brain significantly extracts serine, 220 200 180 160 140 120 isoleucine, leuglutamine, proline, glycine, valine, Va ne, ji.moles/Iiter cine, and lysine from the arterial blood (Table 1). In patients with CR1, exchanges of serine, proline, leucine, and lysine Fig. 2. Relationships between va/me A-V difference and arterial va/inc in 8 subjects with normal renal function (solid circles) across the brain are not different from controls. Conversely, concentration and in 6 patients with chronic renal insufficiency (open circles). glycine and V2cystine uptake significantly increases, valine and isoleucine extraction significantly decreases, glutamine uptake disappears, and a significant ammonia extraction, not observed in normal subjects, becomes evident (Table I). Both in normal indicates that a 1-ii.mole/liter decrease of [serinela and [BCAAIa

conditions and in CR1 there is an excess in the nitrogen is on the average associated respectively with a 0.187- and extracted by the brain of 69.4 9.86 p.moles/liter (P < 0.001) and 53.5 5.25 moles/liter (P < 0.001), respectively. In patients with CR1, there are increases in arterial concentration of proline, glycine, alanine, citrulline, V2cystine, histidine and ammonia, whereas there are decreases of aspartate, serine, valine, and tyrosine (Table I). The A-V difference of glycine shows a linear relationship with its arterial concentration (P < 0,025) (Fig. 1A), and with the ratio of [glycine]a to [neutral AA]a (P < 0.01), mainly [serine]a plus [BCAA]a1 (P < 0.005) (Fig. 1B). The multiple regression equation of the A-V difference of glycine on [glycine]a, [serine]a, and [BCAA]a is: glycine A-V difference = 8.370 + 0.100 [glycineja —0.187 [serine]a —0.024 [BCAA]a, (P < 0.01). The individual regression coefficients are statistically significant (P <0.05, P < 0.001, and P < 0.05, respectively). The equation

0.024-p.mole/liter increase of A-V differences of glycine; more-

over, a l-mole/liter increase of [glycinela is on the average associated with a 0.1 -mole/liter increase of the A-V difference of glycine. The A-V difference of valine is exponentially related to its concentration in arterial blood (P < 0.02) (Fig. 2). The exponen-

tial trend of this relationship cannot be explained. The A-V difference of valine shows a significant relationship with the ratio of [valinela to [neutral AMa (P < 0.02). But, analysis of multiple regression indicates that, in subjects reported here, only the changes of [valine]a markedly affect the variations of the A-V difference of valine. The A-V difference of l/2cystine shows a linear relationship with its arterial concentration (P < 0.01) (Fig. 3A), and with the ratio of [V2cystinela to [neutral AAIa (P < 0.01), mainly [tyro-

sine]a among the others (P < 0.005) (Fig. 3B). The multiple 'BCAA denotes the branched chain amino acids (leucine, isoleucine and valine). The brackets with subscript a signify the arterial concentration.

regression equation of the ¼cystine A-V difference on [(/2cystinela and [tyrosineja is: l/2cystine A-V difference = 4.261 + 0.078 [Y2cystineja —0.121 [tyrosineja, (P < 0.01). The individual

508

Deferrari et a! y = —0.516 + 2.918x (r = 0.765; P <0.005) y= —0.913 + 0.084x(r= 0.687; P< 0.01)

f12

y —17.356 + 0.377x (r = 0.844; P <0.001) 0

+30

(B)

(A) 0

+25

0

0

+20

+10

+15

+4

0

'- +2

.. •o . 30

0

+5

S

70

110

Cystine, pnio/es/Iiter

0.6

1.8

0

3.0

S

Cystine/tyrosine

.

Fig. 3. Relationships between

A-V difference and A arterial to arterial '/2cystine concentration and B the ratio of arterial yrosine concentration in 8 subjects with normal renal function (solid circles) and in 6 patients with chronic renal insufficiency (open circles).

S

30

50

70

90

110

Ammonia, p.mo/es/Iiter

4. Relationships between ammonia A-V difference and arterial ammonia concentration in 8 subjects with normal renal function (solid circles) and in 8 patients with chronic renal insufficiency (open circles). Fig.

regression coefficients are statistically significant (P < 0.05 and

P < 0.05, respectively). The equation demonstrates that a 1p.mole/liter decrease of [tyrosineja is on the average associated

with a 0.121-pmole/liter increase of the A-V difference of

major specific carrier systems for the transport of neutral,

V2cystine; furthermore a l-mole/liter increase of [V2cystine] is on the average associated with a 0.078-ji.mole/liter increase in the A-V difference of Finally, the A-V difference of ammonia is linearly correlated with its arterial concentration (P < 0.001) (Fig. 4).

basic, and acidic AA's [15]. Data obtained in the same animal indicate that changes in blood AA concentrations markedly

Discussion

Findings presented here demonstrate that in normal man, in the postabsorptive state, the brain takes up serine, glutamine, proline, glycine, valine, V2cystine, isoleucine, leucine, and lysine from arterial whole blood. Glutamine and valine uptakes account for 44% of the total AA extraction by the brain. These findings partially confirm previous data obtained from plasma [16] and whole blood measurements [17]. The major discrepan-

cy is the extraction of ¼cystine observed in our study. In

affect the AA entry into the brain [22—251. Results presented here demonstrate that alterations in arterial

AA concentration observed in CR1 affect cerebral uptake of some AA's. Thus, increased arterial glycine induces an increased cerebral uptake of this AA. The cerebral uptake of glycine is also facilitated by the decreased arterial concentration of serine and BCAA, which are likely to be in competition

with glycine for the neutral AA transport system [151. In addition, in the presence of an augmented arterial /2cystine, the brain takes up this AA in larger amounts. Such an uptake is also

increased by the reduced arterial concentration of tyrosine, which probably competes with Y2cystine for the neutral AA

addition, our data demonstrate that the brain extracts glutamine

transport system [15]. Finally, the decreased arterial concentration of valine is the major factor for the low cerebral uptake of

from whole blood, as already shown from plasma measurements [18]. As previously reported [19], in normal conditions there is no significant uptake of ammonia by the brain.

arterial AA concentration, as found in the presence of malnutrition and/or advanced uremia [26, 27], may cause more striking

In CR!, exchanges across the brain are altered for glutamine,

changes of cerebral AA uptake than do those detected in

glycine, valine, Y2cystine, isoleucine, and ammonia. These alterations are observed even in patients with relatively high residual GFR, in good general health, well-nourished, and actively employed. Furthermore, no patient presented obvious neurologic abnormalities. Because cerebral blood flow is nor-

patients studied here. An interesting finding is the cerebral ammonia extraction observed in CR!, as previously reported [281. The appearance of such an extraction is due to the slight increase in arterial ammonia, already suspected in CR1 [10], A similar observation has been reported in patients with liver failure [29]. The lack of cerebral glutamine uptake, previously shown by measuring this AA on plasma [18], is a major change observed in patients with CR!. This defect in cerebral glutamine uptake, in association with ammonia extraction, might be due to an increased synthesis of glutamine by the brain, analogously to that occurring in hepatic [30, 31] and respiratory [32] insufficiency. But, in contrast to what has been observed in these two last conditions [33—35], the ratio of CSF glutamine to plasma glutamine is low in CR! [7] and does not support the hypothesis that an increased production of glutamine by the brain entirely

mal or only slightly increased in patients with CR1 [6, 201, the abnormalities of cerebral AA and ammonia metabolism detect-

ed in CR! cannot be accounted for by variations of cerebral blood flow induced by this condition. Moreover, the magnitude

of the variations (both increase and decrease) of A-V differences of AA's and ammonia observed in CR! may quite unlikely be due to cerebral blood flow changes.

The transport of AA from the arterial blood into the brain is

controlled by three factors: cerebral blood flow, the BBB permeability, and blood concentration of AA [211. In the rat, the AA transport across the BBB takes place through three

this AA. It can be supposed that more severe alterations in

Brain amino acid and ammonia metabolism in rena/failure

accounts for the disappearance of the cerebral uptake of this AA. An impaired utilization of glutamine by the brain is more probable. The low ratio of CSF glutamine to plasma glutamine might suggest a decreased transport of glutamine across the BBB and a consequent skip of cerebral utilization of this AA. But, a defective degradation of glutamine by cerebral tissue, as observed in other organs [10, 11, 18, 361, cannot be ruled out. It

must be pointed out that no correlation between the A-V differences of glutamine and the arterial concentration of gluta-

mine or any other neutral AA was detected in this study Consequently, the defective transport of glutamine into the brain does not seem to be accounted for by the alterations in blood AA concentration observed in CR!. The excess in the nitrogen extracted by the brain both in normal conditions and in patients with CR! suggests that the brain releases similar amounts of nitrogen in compounds not: detectable by the methods used in this study; in fact nitrogen balance across the brain must be at equilibrium. The functional sequel of the alterations in the AA exchanges across the brain observed in CR1 is unknown. These alterations may bring about changes and imbalances to the cerebral content of AA, and brain metabolism may be altered. It is known that

glutamine is a major exogenous source for glutamate and -yaminobutyric acid formation by cerebral cells [37, 38], and that glycine is an important neurotransmitter [39]. Moreover, the BCAA supply to the brain is important for protein, lipid, and glutamate synthesis, and for fuel provision [40—45]; in children. and in rats, isoleucine and valine deficiency produces neurologic symptoms and cerebral lesions [45—47]. Furthermore, cere-

bral ammonia extraction may interfere with cerebral metabolism similarly, even if to a much lesser extent, to that occurring in hepatic insufficiency. In conclusion, the changes of cerebral metabolism observed in patients with CR1 may play a pathogenic role in uremic encephalopathy. Acknowledgments Some of the data reported in this paper appeared in an abstract presented at the 2nd International Congress on Nutrition in Renal Disease, Bologna, Italy, June 13—15, 1979. This study was supported by grants 39/74.00860.65, 42/77.01578.65, and 95/78.02759.65 from the

Consiglio Nazionale delle Ricerche under the cooperative agreement between Italy and the USA. Mr. M. Bruzzone gave technical assistance. Mrs. F. Tincani prepared the manuscript and the figures.

509

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