Citrullinemia

ABSTRACT. Background: The small bowel is believed to play a crucial role in endogenous arginine synthesis. Therefore, an insufficient arginine supply in the situation of massive intestinal resection might impede normal arginine metabolism. This study sought to determine the clinical and metabolic effects of an arginine-free diet in stable short-bowel patients. Methods: Four patients, mean age 49 years (range: 26-67), mean time from intestinal resection 46 m (range: 15-97), and remnant small bowel of 30 to 100 cm consumed an L-amino acid arginine-free diet (egg pattern) for 5 days (0.9 g protein equivalent/kg/d plus malabsorption adjustments). Fasting plasma amino acids, ammonium, and blood chemistries were assessed at days 0, 3, and 5. Urinary orotate, orotidine, uric acid, urea, creatinine, and total nitrogen were evaluated daily. Results: Significant decreases in plasma levels of arginine, ornithine, and hydroxyproline occurred at day 5. A decreasing trend in plasma citrulline and a significant plasma glutamine increase were also observed in the same period. Conversely, ammonium concentrations remained normal. Regarding urine compounds, striking orotic aciduria with a peak at day 4 (14-fold vs baseline) and significant decreases in uric acid and urea excretion were found. There were no relevant clinical events. Conclusions: Despite the limited number of patients in our work and their relative heterogeneity, our results support the idea of the indispensability of an exogenous arginine supply in humans under short bowel syndrome conditions. Studies in larger series are needed to further investigate these findings. (Journal of Parenteral and Enteral Nutrition 28:315-323, 2004)

The fact that arginine may become an essential amino acid under short bowel syndrome has led us to draw the following conclusions for patients with this condition: first, and most important, parenteral nutrition (PN) formulas lacking in arginine (ie, classic renal formulas) should be avoided; second, an adequate exogenous arginine supply should be assured under all circumstances; and third, special attention should be paid to arginine supply when there are other coexisting arginine deficiency risk conditions, such as sepsis, trauma, burns or catch-up growth.

Background

Since the study by Rose et al1 in the early fifties, arginine is assumed to be a dispensable amino acid in humans. Later on, Carey et al,2 assessing urinary orotate excretion, amino acid plasma profiles, and blood ammonia levels in 5 healthy subjects consuming an arginine-free diet during 6 days, found only a slight decrease in postabsorptive plasma arginine levels. In a tracer study using stable isotopes in 8 healthy men, Castillo et al3 showed that de novo arginine synthesis rates were unaffected by a 6-day arginine-free diet, providing further evidence for arginine dispensability in humans. Nevertheless, according to recent studies in humans,4-6 it can be argued that the metabolic demands for arginine can exceed the biosynthetic capacity of the organism under specific physiologic or pathologic circumstances. Because of this fact, arginine is considered a conditionally indispensable amino acid.7

Several interesting findings on intermediate ureacycle metabolism have come from the work of Wakabayashi and Jones8 with the discovery of [Delta]^sup 1^-pyrroline-5-carboxylate synthase (5-PCS), a bifunctional enzyme that catalyzes the reduction of glutamate to [Delta]^sup 1^pyrroline-5-carboxylate (P5C), a critical step in the biosynthesis of proline and ornitbine, which is further converted to citrulline and arginine (Figs. 1, 2). The latter author also discovered the exclusive production of 5-PCS in the small bowel of rats10 and, more recently, in humans (Wakabayashi, personal communication, A.S.P.E.N. 2000). Working with these findings, in 1995 the same author found a clinical and biochemical picture of arginine deficiency in rats undergoing 80% fixed small bowel resection and fed an arginine-free diet.11 The most relevant discoveries from this study were weight loss, negative nitrogen balance, drastically reduced concentrations of arginine (especially in skeletal muscle), moderate reductions in plasma citrulline, increases in plasma and muscle levels of glutamine, and high excretion of orotic acid in urine.

Short bowel syndrome (SBS) is a state of severe malabsorption in humans, resulting from absence or removal of a large percentage of the small bowel (>50%). Hyperammonemia, orotic aciduria, and severe neurologic symptoms have been reported in SBS patients treated with PN lacking in arginine,12-14 which seems to express a need for the substrate in this particular condition. Additionally, Crenn et al15 found a strong correlation between citrullinemia and small bowel length in 57 SBS individuals, confirming the findings in resected rats.

According to previous reports in animals and humans and the crucial importance of the intestine in the synthesis of citrulline, which in turn would be converted to arginine by the kidney, we hypothesized that patients with massive intestinal resection would have strongly impaired endogenous arginine synthesis. To test this idea, we designed a study aimed toward assessing the metabolic and clinical consequences of a short period on an arginine-free diet in patients with short-bowel syndrome. Among the most relevant biochemical results we expected to find were orotic aciduria without hyperammonemia and decreased plasma concentrations of amino acids such as arginine, ornithine, and citrulline, with increased glutamine.

MATERIALS AND METHODS

Patients

The characteristics of the patients are described in Table I. Four stable SBS volunteers (2 men and 2 women) with a mean age of 49 years (range: 26-67) were recruited between April 1996 and May 2000. The mean time from the main intestinal resection was 46 months (range: 15-97) and the mean length of remnant small bowel was 67 cm (range: 30-100). All resections were performed for intestinal ischemia because of the following conditions: case 1, a huge benign uterine tumor; case 2, mesenteric venous thrombosis; case 3, mesenteric arterial thrombosis; and case 4, mesenteric trauma (car accident). All subjects were stable in terms of nutrition parameters and clinical condition; free from active cancer, infectious disease, and severe organ failure (liver, renal, cardiac, or lung failure); and had undergone their last surgical procedure more than 6 months before starting the study.

The study protocol was approved by the Ethical Committee of our hospital according to the Helsinki Declaration of 1964, as revised in 1989. Written consent was obtained from all participants after the purpose of the study and the potential risks involved had been fully explained to each subject. The conditions for withdrawal from the study included the following: (1) plasma ammonium levels twice the upper normal limit; (2) development of neurologic symptoms (except moderate headache or some degree of fatigue); (3) unexpected onset of any other disease; and (4) voluntary withdrawal.

Plasma ammonium and amino acids and urinary orotic acid excretion were also assessed in a control group formed by 28 healthy staff volunteers (50% males 46 years old; range: 26-64) for use as reference values.

The patients' associated diseases and treatment received were as follows:

(a) case 1 (woman, 66 years). Obesity, hypertension, and diabetes type II previous to intestinal resection. After resection, obesity persisted and the patient experienced some urinary infections. At entry into the study, urinary cultures were negative.

(b) Case 2 (man, 26 years). No associated or systemic diseases related to venous thrombosis.

(c) Case 3 (man, 55 years). Buerger's disease, which led to arterial bypasses and supracondylar amputation of 1 leg. He also had mild renal failure (plasma creatinine: 130-140 µmol/L; proteinuria 2.5-3 g/d) because of glomerular angiosclerosis.

(d) Case 4 (woman, 50 years). No previous pathologies. In December 1995, 4 consecutive abdominal surgeries after a traffic accident resulted in a remnant bowel of 100 cm of jejunum with a jejunostomy and one-half colon excluded (reanastomosed 8 months later). She also had 3 subocclusive episodes in 4 years, no surgery required. She experienced prominent watery diarrhea (7 to 8 stool movements/d), with frequent hypokalemia (malabsorption of bile salts detected).

Patient treatments during the study period are shown in Table II. PN in patient 1 was interrupted 2 days before the nutrition intervention and switched to daily IV glucose (200 g/d) and fluids with saline perfusion up to 4500 mL. The previous PN formula was as follows: nitrogen 36 g/week (Vamin 14; Pharmacia, Stockholm, Sweden), lipids 50 g/week (Intralipid; Pharmacia) and dextrose 800 g/week. Micronutrients and electrolytes were also supplied. No extra choline was supplied to any patient during the study.

Study Design

All patients were referred to the hospital Endocrinology Service the night before beginning the experimental diet and discharged after blood collection at day 5. All subjects were given the experimental diet for 5 complete days. The dietitian in charge supervised all the meals.

Study Diet

Study diet characteristics are displayed in Table III. For each patient, dietary assessment was performed by a trained dietitian before the beginning of the study (AF) in order to evaluate the usual intake of energy, macronutrients, and taste preferences. Also, a single meal with the study diet was consumed before the basal day to assure acceptance.

The arginine-free diet was composed of 5 meals per day during 5 days, with the same amount of amino acids per meal. All patients continued their usual vitamin and mineral preparations as before the study period.

Daily energy and protein requirements for each patient were calculated on an individual basis with the following approach:

(a) Energy Requirements. The daily energy intake was designed to maintain initial body weight and was kept constant over the entire study. The Harris-Benedict equation was used to determine the basal metabolic rate (BMR); adjusted weight was used in case 1 owing to obesity. Total daily energy requirements were calculated by adding to the BMR extra energy for physical activity, dietary-induced thermogenesis, and fecal losses because of malabsorption (30% in cases 2 and 4, 60% in case 3, and 80% in case 1 of energy intake losses) as reported elsewhere.16,17

Nonprotein energy distribution consisted of 30% to 40% of energy supply from fat and 60% to 70% from carbohydrates (CHO). CHO sources were protein-free products such as cookies, macaroni, spaghetti, and bread (Loprofm; Nutricia, Zoetermeer, Holland), and regular sugar, honey, jelly, and fruit, such as bananas and apples. The main fat energy sources were margarine and olive oil.

(b) Protein Requirements. Nitrogen equivalent to 0.9 g protein (N × 6.25) kg^sup -1^ day^sup -1^ was provided, plus an extra amount to compensate for malabsorption losses16,17 (30% in cases 2 and 4; 50% in cases 1 and 3). The L-amino acid mixture3 had a hen's egg amino acid profile excluding arginine, which was totally removed from the mixture (Table IV). In order to compensate for nitrogen losses caused by arginine exclusion, hen's egg nonessential amino acid proportions of glycine and proline were increased by 488% and 53%, and aspartate, glutamate, and serine were decreased by 28%, 25%, and 73%, respectively. The mixture was prepared by SHS Laboratories (Liverpool, UK) and mixed with a flavouring agent (Modjul Flavour System; SHS) and sucrose to improve taste (100 g of amino acids per 2.5 g of flavoring agent and 25 g of sucrose). Only water ad libitum was allowed to cover fluid needs.

Physical Exploration

The Mini Mental Test and an ECG were performed in each patient on the morning of the basal day and at the end of the study period. Weight, blood pressure, temperature, urine volume, number of stool movements, and heart rate were recorded daily.

Blood and Urine Assays

Overnight fasting plasma amino acids and ammonia, and serum biochemical analytes including liver function tests, albumin, urea, creatinine, electrolytes, urates, triglycerides, and total cholesterol were determined after standard procedures at baseline and on days 3 and 5. Serum urea and creatinine were also assessed on day 4. Hematologic count was measured at baseline and on days 3 and 5.

Two milliliters of blood were drawn, placed in glass tubes containing EDTA, and rapidly kept in an ice bath. Samples were centrifuged at 4°C and assayed immediately for plasma ammonium concentration by a quantitative enzymatic method with a Centrifugal Cobas Fara II Autoanalyzer (Bio-Merieux).

For plasma amino acid assessment, 6 mL of blood were drawn, placed in EDTA-containing Vacutainer glass tubes, and centrifuged for 15 minutes at 2000 × g and 4°C. Aliquots of plasma were deproteinised with ultrafiltration tubes (Ultrafree-MC; Millipore) and stored at -80°C until amino acid analysis. Quantitative analyses of amino acids in plasma were performed by precolumn derivation with phenylisothiocyanate by reverse phase high-performance liquid chromatography (HPLC) in Waters equipment with a digital computer, according to the Waters PICO-TAG procedure.18

Daily 24-hour urine output was collected for each subject, frozen, and stored. Volume was recorded. The following substances were assayed in urine: orotic acid (orotate), orotidine, creatinine, urea, urate, and total nitrogen. Orotic acid and orotidine excretion were measured after heating 24-hour urine samples to 70°C to assure total solubility; the analysis was carried out by anion-exchange HPLC as described by Brusilow and Hauser19 and modified by Arranz et al.20 Total nitrogen was determined by the Kjeldhal procedure.

Statistical Analysis

Continuous variables are presented as means, SDs, and 95% confidence intervals. Nonparametric repeated measures comparisons were performed by Friedman's test. A p value

RESULTS

Clinical Results

Diet Tolerance. The diet was generally well tolerated, although patients complained that it was monotonous.

Neurologic Exploration. The Mini Mental test was normal at entry and at the end of the study in all cases. Mild headache episodes appeared in cases 2, 3, and 4 for a few hours in the middle of the study period. No treatment was required. Mild fatigue was noted after initiation of the study diet in 3 of the patients (cases 2, 3, and 4), especially from day 2 on. Appetite was preserved in all patients, and thirst increased in case 4 (last 2 days).

Body Weight. In all patients but 1, there were no significant body weight changes during the study period. In case 2, body weight decreased by 1 kg.

Urine and Feces. No variations in urine output were observed. In cases 1, 2, and 3, there were no increases in stool movements or jejunostomy output. Case 4 showed increased stool movements from 7 to 8 per day at baseline to 10 to 12 per day in the last 2 days.

Blood Pressure. Daily records of blood pressure were normal.

Heart Rate. No alterations were detected.

Biochemical Results

Blood Chemistry. No relevant fluctuations were observed over the study period, and levels of the following parameters remained within normal values: total cholesterol, triglycerides, liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], gamma-glutamyltransferase [[gamma]-GT]), total bilirubin, glucose, albumin and sodium. Uric acid levels decreased between 13% and 43%. Creatinine levels were normal and remained unchanged in all patients but 1 (case 3), in whom levels decreased from 146 µmol/L to 110 µmol/L at day 5. Urea levels were normal and decreased over the study except in case 4 (from 3.6 mmol/L to 4.1 mmol/L at day 5). Potassium levels showed no alterations in 3 of the patients, but in case 4 (with severe diarrhea), levels declined from 3.62 to 2.99 mmol/L at day 5.

Hematologic Parameters. No relevant changes occurred in hemoglobin, hematocrit, total leukocytes, or platelets.

Fasting Plasma Ammonium. Plasma ammonium concentrations were within normal limits (100 µmol/L).

Plasma Amino Acid Levels. Arginine, ornithine, and hydroxyproline plasma concentrations decreased significantly from baseline to day 5 in all patients (Table V). Ornithine basal values were lower than controls in cases 2 and 3 (41 and 42 µmol/L, respectively) and were similar to controls in the other 2 patients (65 and 100 µmol/L, cases 1 and 4). At day 5, all patients showed lower ornithine concentrations (28 ± 2 µmol/L) than the control group (89 ± 28 µmol/L), and there was a marked reduction of 24% to 78% relative to the basal values (mean reduction: 34 ± 31 µmol/L). At day 3, levels were slightly lower than at day 5 in 3 of the cases. Basal arginine values were normal in 3 patients (63, 81, and 83 µmol/L) and elevated in case 1 (142 µmol/L) compared with controls (59 ± 25 µmol/L). At day 5, plasma arginine had decreased in all the patients between 29% and 78%, with a mean of 55 µmol/L. Values at day 3 were lower than at basal analysis in 3 patients. In 2 cases, arginine showed a slight increase at day 5 with respect to day 3.

Basal citrulline values were lower than controls in cases 1 and 2 (20 and 12 µmol/L) and were similar to controls in cases 4 and 3 (33 and 31 µmol/L), the latter patient having mild renal failure. In all patients but 1, citrulline values dropped at day 5 from 10% to 50% (mean reduction: 3.7 ± 6.3 µmol/L). Plasma citrulline concentrations in case 3 remained high at day 5, possibly owing to the renal insufficiency.

Plasma glutamine levels at baseline in the patient group were within the reference range except for case 3, with lower values (380 µmol/L). After 5 days of arginine deprivation, a mean significant statistical increase of 162 ± µmol/L was observed in cases 1, 2, and 3 (6.6%, 23.1%, and 82.1%, respectively). In contrast, glutamine values in case 4 fell unexpectedly by almost 50%.

Initially, plasma hydroxyproline concentrations were within the reference values in all cases. Day 5 hydroxyproline levels were 24% to 69% lower than baseline concentrations (9.0 ± 2.1 vs 16.2 ± 1.3 µmol/L), but only 1 patient (case 4) exhibited levels below reference values (17 ± 10 µmol/L).

With respect to plasma threonine, glutamate, aspartic acid, serine, asparagine glycine, taurine, histidine, alanine, proline, [alpha]-aminobutyrate, tyrosine, aspartate, valine, leucine, isoleucine, cysteine, methionine, phenylalanine, and lysine, no alterations were found.

Urinary Nitrogen Compounds. Various alterations in urinary excretion were observed in some of the assessed compounds (Table VI). At baseline, orotic acid (orotate) urine excretion values (0.44 ± 0.07 mmol/mol creatinine) were comparable to the reference values (0.60 ± 0.42 mmol/mol creatinine). Varying increments of orotic acid excretion occurred between baseline and days 1 to 5, particularly in cases 1, 2, and 3 (Fig. 3). These elevations cannot be explained by the mild changes observed in creatinine urinary excretion. The highest values occurred in case 2 (15.3 mmol/mol creatinine at day 4) and case 3 (15 mmol/mol creatinine at day 1) and represented 30 and 60 times basal levels. These values were, of course, strikingly abnormal, only seen in urea cycle disorders in humans27 and arginine deficiency in animals.22-24 At day 5, orotic acid excretion values were 4.8-, 15.2-, 6.1-, and 1.6-fold greater than baseline concentrations, yet lower than those observed at day 4. With respect to case 4, orotate levels at days 3 and 4 were twice basal values but still within normal. Overall, orotic acid excretion levels in urine increased significantly from day 0 to day 5.

Baseline orotidine excretion was within the reference values (0.23 ± 0.16 mmol/mol creatinine) in cases 2, 3, and 4, and higher than controls in case 1 (1.2 mmoL/moL creatinine. Orotidine excretion values showed almost no variation in 3 of the patients and doubled in case 2.

Urinary urea excretion relative to basal at day 5 was decreased in cases 1, 2, and 3 (39%, 19%, and 41%, respectively). We found an increase (50%) at day 5 vs day 0 only in case 4. Overall, urea excretion decreased significantly from day 0 to day 5. Urinary nitrogen excretion followed a similar trend in all the patients.

Urinary creatinine excretion showed slight decreases of 6% to 16% in all patients from day 0 to day 5. Urate excretion fell us day 0 with statistical significance in all cases, being 70% in case 2, 50% in case 1 and 3, and 20% in case 4.

Changes in urinary nitrogen excretion followed a pattern similar to that of urea. After 5 days of arginine-free diet, values decreased by 30%, 10%, and 40% in cases 1, 2, and 3, respectively, but increased by 60% in case 4.

Diuresis remained fairly constant in all patients.

DISCUSSION

This is the first study investigating the effects of an arginine-free diet in individuals with SBS. With reference to the length of the study, we chose 5 days instead of the 6 to 8 days usually established in studies of amino acid essentiality because of the primacy for the safety and comfort of the patient and the conviction that this period would be long enough to detect possible metabolic alterations.

Several authors25,26 state that arginine needs in humans are met in the following ways: (1) dietary exogenous supply; (2) turnover of body proteins; (3) intestinal synthesis of citrulline as a precursor, which is later converted into arginine by the kidney; and (4) to a minimum extent, arginine regeneration in other tissues, such as brain or lymphocytes, through NO-synthase activity, and possibly intestinal synthesis of arginine from proline, as in young pigs.

Though it remains uncertain which of the abovementioned mechanisms contributes most to endogenous arginine production, studies in humans27,28 point to renal citrulline uptake as an important factor related to renal arginine synthesis, as occurs in rats.29 Citrulline produced in the small intestine is released to the circulation and taken up by the kidney, where it is converted to arginine in an equimolar fashion (gutkidney axis). Hence, it seems reasonable to assume that citrulline synthesis would be decreased in patients with a considerably lower enterocyte mass, as occurs in SBS patients.

Orotic acid is an intermediate in the pathway of pyrimidine synthesis, and in healthy subjects it is excreted in minimum quantities.30 Increased orotic acid excretion has been proposed to result from decreased urea cycle capacity because of arginine deficiency or urea-cycle congenital disorders.21 Also, mild orotic aciduria has been found in severe trauma victims31 and pregnancy.32 The reason for orotic aciduria in states of arginine deficiency, at least in rats, appears to be associated with the increase of plasma and hepatic glutamine concentrations which, in turn, activate cytosolic enzyme carbamyl phosphate synthetase II used to generate orotate (instead of mitochondrial carbamyl phosphate synthetase I).33 As previously stated, some case reports have shown increases in plasma orotic acid concentration (orotic aciduria was not determined) and hyperammonemia in patients with intestinal resection and chronic renal failure receiving arginine-free total PN (TPN) solutions.12-14 This supports the idea that, in addition to other parameters, urinary orotic acid determination could be used as a biochemical marker of arginine deficiency in SBS. Carey et al2 did not observe an increase in orotic acid excretion in healthy individuals after 6 days of arginine-free diet. Hence, the dramatic increase in orotic acid excretion found in our SBS patients under exogenous arginine deprivation has no other feasible explanation than an arginine deficiency in SBS individuals compared with healthy ones. This deficiency may be explained by reduced endogenous arginine synthesis caused by the smaller citrulline-producing enterocyte mass. The lesser increase in orotic acid excretion observed in 1 of our patients (case 4) might be partially explained by the dehydration that occurred during the study period.

Orotic aciduria was not accompanied by hyperammonemia in our study. Yet it cannot be assured that blood ammonium would not have increased after a few more days of arginine deprivation (6-7 days more), as described in the aforementioned case reports involving SBS patients with renal failure fed arginine-free PN.12-14 It is possible that the absence of hyperammonemia with marked orotic aciduria may be a result of partial conversion of dietary proline to ornithine in the residual intestine, which prevented hyperammonemia but did not suffice to avoid mild orotic aciduria.

The descent of plasma uric acid levels in all of our patients was also described in resected rats fed without arginine.11 This fall might be the result of an elevated synthesis of pyrimidine nucleotides and a reduced synthesis of purine nucleotides in arginine-deficient circumstances, probably as a consequence of ammonium preference for the pyrimidine pathway. Another explanation might be the absence of purine precursors in our formula; in TPN patients, uric acid levels were similarly decreased in both urine and plasma.34

The mild plasma urea decline we observed in cases 1, 2, and 3 (case 4 experienced dehydration) contrasts with the results of the aforementioned study in resected rats11 fed an arginine-free diet for 4 weeks in which a rise in blood urea was observed. A possibly lower nitrogen intake compared with previous amounts, a limited absorption capacity of amino acid mixtures in SBS individuals, or the longer duration of the experimental study under arginine-devoid conditions might explain the differing trends in urea excretion seen in the 2 studies. Additionally, the decrease in plasma urea in 3 of our patients was accompanied by a fall in urinary excretion.

Changes in plasma and tissue amino acids have been described in arginine-deficient animals; these include falls in plasma arginine, ornithine, and citrulline and rises in plasma glutamine, alanine, and [gamma]-amino butyric acid. As observed in animal models,11,33,35 our 4 SBS patients experienced rapid and substantial changes in blood urea cycle-related amino acids after 5 days of arginine deprivation. After 3 hours of fasting, a single meal of arginine-free diet led arginine-dependent young ferrets to significant falls in plasma arginine and ornithine, and a relevant rise in glutamine and ammonium.36 Wakabayashi et al11 found that arginine-free diets significantly affected arginine, ornithine, and glutamine plasma concentrations in resected rats; the animals developed arginine deficiency and lost weight.

Fasting plasma levels of arginine in our patients fell from baseline values. In arginine-dependent animals,36,37 including carnivorous and some newborn omnivorous species, fasting and fed plasma arginine concentrations are one of the most sensitive indicators of arginine status, with a dramatic plasma fall occurring in some after only a few hours of arginine withdrawal. Mean ornithine plasma values descended in all of our SBS patients (baseline: 62 ± 14; day 5: 28 ± 2 µmol/L), reaching levels below normal (89 ± 28 µmol/L). Ornithine is an amino acid of endogenous origin tightly linked with arginine metabolism. Plasma ornithine decreases have been reported in arginine-dependent animals lacking an exogenous arginine supply, with ornithine levels acting as a possible indicator of arginine deficiency.36 Moreover, arginine supplementation in humans by oral or parenteral routes leads to significant increases in plasma ornithine.38-40 Using stable isotope tracer techniques in healthy humans with restricted arginine intake, Castillo et al41 found a significant reduction in the flux and oxidation rate of ornithine.

It is widely accepted that the bowel is the major producer and contributor to the citrulline body-pool and plasma levels. Citrulline is synthesized from glutamine in the small intestine and it is also produced in the liver, where it stays for endogenous purposes (urea cycle). In agreement with Crenn et al,42 who observed a significant correlation between remnant small bowel length and plasma citrulline concentrations in humans (r = .83, p

A novel but not unexpected finding in this study is the descent of circulating hydroxyproline in all patients. Barbul et al46 observed in 1990 an increase in the amount of hydroxyproline deposited within a subcutaneously placed catheter in healthy subjects after high oral doses of arginine during 15 days, suggesting a relationship between arginine supply and augmented hydroxyproline synthesis. On the other hand, proline concentrations were not affected by arginine removal, as in study with resected rats.11

Relevant considerations as to the fate of proline in the formulas we used are important: (a) small intestine in the only site where glutamate (from glutamine or [alpha]-Ketoglutarate), P5C, ornithine, and proline are interconverted (there is no doubt that is enteral conversion from proline to ornithine via P5C); (b) therefore, orally administered proline but not parenterally administered proline could have been converted to ornithine, citrulline, and arginine, as was demonstrated in neonatal pigs.47,48 At present, we have no information on the activity of proline oxidase in the human intestine. In the pig model, however, small bowel proline oxidase activity markedly decreased with age.49

Despite the limited number of patients in our study and their relative heterogeneity, our hypothesis is supported by the biochemical findings, including the absence of hyperammonemia and rise in orotic acid excretion in urine and the fall in plasma levels of urea-cycle amino acids arginine and ornithine, with a trend to decreased citrulline and a rise in glutamine. All these data support the idea of the indispensability of an exogenous arginine supply in humans under SBS conditions, as reported in resected rats. Studies in larger series are needed to further investigate these findings. Nevertheless, we believe that attention should be paid to the arginine supply provided to SBS patients, particularly those with concomitant severe trauma, sepsis, burns, or renal failure, and that the usual arginine-lacking renal formula should be avoided.

ACKNOWLEDGMENTS

The amino acid formula was kindly provided by SHS International Ltd. The technical assistance of Monica Bullo, Laura Gabaso, and Ferran Balduille and the linguistic advice of Celine Cavallo were greatly appreciated.

REFERENCES

1. Rose WC, Haines WJ, Warner DT. The amino acid requirements of man, V: the role of lysine, arginine, and tryptophan. J Biol Chem. 1954;206:421-430.

2. Carey GP, Kime Z, Rogers QR, et al. An arginine-deficient diet in humans does not evoke hyperammonemia or orotic aciduria. J Nutr. 1987;117:1734-1739.

3. Castillo L, Ajami A, Branch S, et al. Plasma arginine kinetics in adult man: response to an arginine-free diet. Metabolism. 1994; 43:114-122.

4. Suh H, Peresleni T, Wadhwa N, McNurlan M, Garlick P, Goligorsky MS. Amino acid profile and nitric oxide pathway in patients on continuous ambulatory peritoneal dialysis: L-arginine depletion in acute peritonitis. Am J Kid Dis. 1997;29:712-719.

5. Zamora SA, Amin HJ, McMillan DD, et al. Plasma L-arginine concentration, oxygenation index, and systemic blood pressure in premature infants. Crit Care Med. 1998;26:1271-1276.

6. Yu H-M, Ryan CM, Burke JF, Tompkins RG, Young VR. Relations among arginine, citrulline, ornithine and leucine kinetics in adult burn patients. Am J Clin Nutr. 1995;62:960-968.

7. Reeds P, Schaafsma G, Tome D, Young V. Criteria and significance of dietary protein sources in humans: summary of the workshop with recommendations. J Nutr. 2000; 130:18748-1876S.

8. Wakabayashi Y, Jones MB. Pyrroline-5-carboxylate synthesis from glutamate by rat intestinal mucosa. J Biol Chem. 1983;258: 3865-3872.

9. Wakabayashi Y, Yamada E, Yoshida T, Takahashi H. Arginine becomes and essential amino acid after massive resection of rat small intestine. J Biol Chem. 1994;269:32667-32671.

10. Wakabayashi Y, Yamada E, Hasegawa T, Yamada R. Enzymological evidence for the indispensability of small intestine in the synthesis of arginine from glutamate, I: pyrroline-5-carboxylate synthase. Arch Biochem Biophys. 1991;291:1-8.

11. Wakabayashi Y, Yamada E, Yoshida T, Takahashi N. Effect of intestinal resection and arginine-free diet on rat physiology. Am J Physiol. 1995;269:G313-G318.

12. Yamada E, Wakabayashi Y, Saito A, Yoda K, Tanaka Y, Miyazaki M. Hyperammonaemia caused by essential amino acid supplements in patient with short bowel. Lancet. 1993;341: 1542-1543.

13. Grazer RE, Sutton JM, Friedstrom S, McBarron FD. Hyperammonemic encephalopathy due to essential amino acid hyperalimentation. Arch Intern Med. 1984;144:2278-2279.

14. Nakasaki H, Katayama T, Yokoyama S, et al. Complication of parenteral nutrition composed of essential amino acids and histidine in adults with renal failure. JPEN J Parenter Enteral Nutr. 1993;17:86-90.

15. Crenn P, Coudray-Lucas C, Thuillier F, Cynober L, Messing B. Postabsorptive plasma citrulline concentration is a marker of absorptive enterocyte mass and intestinal failure in humans. Gastroenterology. 2000;119:1496-1505.

16. Messing B, Pigot F, Rongier M, Morin MC, Ndeindoum U, Rambaud JC. Intestinal absorption of free oral hyperalimentation in the very short bowel syndrome. Gastroenterology. 1991;100: 1502-1508.

17. Woolf GM, Miller C, Kurian R, Jeejeebhoy KN. Nutritional absorption in short bowel syndrome: evaluation of fluid, calorie, and divalent cation requirements. Dig Dis Sci. 1987;32:8-15.

18. Heinrikson RL, Meredith SC. Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate. Anal Biochem. 1984;136: 65-74.

19. Brusilow SW, Hauser E. Simple method of measurement of orotate and orotidine in urine. J Chromatogr. 1989;493:388-391.

20. Arranz JA, Riudor E, Rodes M, et al. Optimization of allopurinol challenge: sample purification, protein intake control, and the use of orotidine response as a discriminative variable improve performance of the test for diagnosing ornithine carbamoyltransferase deficiency, Clin Chem. 1999;45:995-1001.

21. Steiner RD, Cederbaum SD. Laboratory evaluation of urea cycle disorders. J. Pediatr. 2001;138(suppl 1):S21-S29.

22. Visek WJ. Arginine needs, physiological state and usual diets: a reevaluation. J Nutr. 1986;116:36-46.

23. Ha YH, Milner JA, Corbin JE. Arginine requirements in immature dogs. J Nutr. 1978;108:203-210.

24. Hartman WJ, Prior RL. Dietary arginine deficiency alters flux of glutamine and urea cycle intermediates across the portaldrained viscera and liver of rats. J Nutr. 1992;122:1472-1482.

25. Young VR, Yu YM. Protein and amino acid metabolism. In: Fischer JE, ed. Nutrition and Metabolism in the Surgical Patient. New York, NY: Little, Brown & Company; 1996:159-201.

26. Wu G, Morris SM. Arginine metabolism in mammals. In: Cynober LA, ed. Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition. Boca Raton, FL: CRC Press; 2004:153-167.

27. Rabier D, Kamoun P. Metabolism of citrulline in man. Amino Acids. 1995;9:299-316.

28. Tizianello A, De Ferrari G, Garibotto G, Gurreri G, Robaudo C. Renal metabolism of amino acids and ammonia in subjects with normal renal function and in patients with chronic renal insufficiency. J Clin Invest. 1980;65:1162-1173.

29. Dhanakoti SN, Brosnan JT, Herzberg GR, Brosnan ME. Renal arginine synthesis: studies in vitro and in vivo. Am J Physiol. 1990;259:E437-E442.

30. Sumi S, Kidouchi K, Imaeda M, Asai M, Ito T, Wada Y. Urinary orotic acid in healthy adults and patients with various diseases. Clin Chim Acta. 1997;266:195-197.

31. Jeevanandam M, Hsu YC, Ramias L, Schiller WR. Mild orotic aciduria and uricosuria in severe trauma victims. Am J Clin Nutr. 1991;53:1242-1248.

32. Glasgow AM, Larsen JW. Urinary orotic acid and pregnancy. Am J Obstet Gynecol. 1984;149:464-465.

33. Gross KL, Hartman WJ, Ronnemberg A, Prior RL. Arginine-deficient diets alter plasma and tissue amino acid in young and aged rats. J Nutr. 1991;121:1591-1599.

34. Koretz RL. Hypouricemia-a transient biochemical phenomenon of total parenteral nutrition. Am J Clin Nutr. 1981;34:2493-2498.

35. Dejong CHC, Welters CFM, Deutz NEP, Heineman E, Soeters PB. Renal arginine metabolism in fasted rats with subacute short bowel syndrome. Clin Sd. 1998;95:409-418.

36. Deshmukh DR, Sarnaik AP, Mukhopadhyay A, et al. Effect of arginine-free diet on plasma and tissue amino acids in young and adult ferrets. J Nutr Biochem. 1991;2:72-78.

37. Flynn NE, Knabe DA, Mallick BK, Wu G. Postnatal changes of plasma amino acids in suckling pigs. J Anim Sci. 2000;78:2369 2375.

38. Hurson M, Regan MC, Kirk SJ, Wasserkrug HL, Barbul A. Metabolic effects of arginine in a healthy elderly population. JPEN J Parenter Enterai Nutr. 1995; 19:227-230.

39. Kelly BS, Alexander JW, Dreyer D, et al. Oral arginine improves blood pressure in renal transplant and hemodialysis patients. JPEN J Parenter Enteral Nutr. 2001;25:194-202.

40. Sigal RK, Shou J, DaIy JM. Parenteral arginine infusion in humans: nutrient substrate or pharmacological agent? JPEN J Parenter Enterai Nutr. 1992;16:423-428.

41. Castillo L, Sanchez M, Chapman TE, Ajami A, Burke JF, Young VR. The plasma flux and oxidation rate of ornithine adaptively declined with restricted arginine intake. Proc Natl Acad Sd USA. 1994;91:6393-6397.

42. Crenn P, Coudray-Lucas C, Thuillier F, Cynober L, Messing B. Postabsorptive plasma citrulline concentration is a marker of absorptive enterocyte mass and intestinal failure in humans. Gastroenterology. 2000;119:1496-1505.

43. Wasa M, Takagi Y, Sando K, Harada T, Okada A. A long-term outcome of short bowel syndrome in adult and pediatrie patients. JPEN J Parenter Enterai Nutr. 1999;23:S110-S112.

44. Pita AM, Wakabayashi Y, Fernandez-Bustos MA, et al. Plasma urea-cycle-related amino acids, ammonium levels, and urinary orotic acid excretion in short-bowel patients managed with an oral diet. Clin Nutr. 2003;22:93-98.

45. Ceballos I, Chauveau P, Guerin V, et al. Early alterations of plasma free amino acids in chronic renal failure. Clin Chim Acta. 1990;188:101-108.

46. Barbul A, Lazarou SA, Efron DT, Wasserkrug HL, Efron G. Arginine enhances wound healing and lymphocyte immune responses in humans. Surgery. 1990;108:331-337.

47. Brunton JA, Bertolo RFP, Pencharz PB, Ball RO. Proline ameliorates arginine deficiency during enterai but not parenteral feeding in neonatal piglets. Am J Physiol Endocrinol Metab. 1999;277: E223-E231.

48. Bertolo RFP, Brunton JA, Pencharz PB, Ball RO. Arginine, ornithine, and proline interconversion is dependent on small intestinal metabolism in neonatal pigs. Am J Physiol Endocrinol Metab. 2003;284:E915-E922.

49. Wu G. Synthesis of citrulline and arginine from proline in enterocytes of postnatal pigs. Am J Physiol. 1997;272:G1382-G1390.

Ana M. Pita, MD*; Angeles Fernandez-Bustos, RD*; Margarita Rodes, PhD[dagger]; Jose A. Arranz, PhD[double dagger]; Cesar Fisac, RD*; Nuria Virgili, MD*; Joan Soler, MD*; and Yasuo Wakabayashi, MD§

From the * Department of Endocrinology and Nutrition, Hospital de Bellvitge, L'Hospitalet de Llobregat, Spain; [dagger]Institut Bioquimica Clinica, Corporacio Sanitaria Clinic, Barcelona, Spain; [double dagger]Laboratorio de Metabolopatias, Hospital Materno-Infantil, Vall d'Hebron, Barcelona, Spain; § Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto, Japan

Received for publication March 25, 2003.

Accepted for publication May 12, 2004.

Correspondence: Ana Maria Pita, Department of Endocrinology and Nutrition, Hospital de Bellvitge, Galle Peixa Llarga s/n, 08907 L'Hospitalet de Llobregat, Spain.

Copyright American Society for Parenteral and Enteral Nutrition Sep/Oct 2004
Provided by ProQuest Information and Learning Company. All rights Reserved