From the Clinical Laboratory and the Department of Anaesthesiology, S:t Erika Sjukhus, Stockholm and the Clinical Laboratory, Sahlgrenska Sjukhuset, Goteborg

Parenteral nutrition by a solution of crystalline amino acids

BY

PETER FURST, Bo HALLGREN, BERTIL JOSEPHSON and ERIK VINNARS

It is a common experience that solutions of hydrolyzed or digested casein ad- ministered intravenously to patients for amino acid nutrition, as first suggested by Elman & Weiner (4), may incidental- ly cause nausea, vomiting and other discomforts. Such symptoms can be provoked in almost any subject by in- creasing the volume or rate of the hydro- lysate infusion. It does not seem prob- able that the amino acids are respon- sible for these symptoms, even if some of them may be toxic when given in large amount. Solutions of pure 1-amino acids have therefore been suggested for par- enteral nitrogen nutrition, with the in- tention of avoiding disagreeable side-ef- fects. Since the essential amino acids have recently become available at a reasonable price, some of these solutions are now on the market. However, it is not yet established that the composition of these solutions is optimal with respect to the nitrogen balance in man. For this reason, we have studied some amino acid patterns in an attempt to suggest a so- lution giving a positive nitrogen bal-

ance, without causing discomfort or other side-effects to the patients.

After several attempts, not reported here, we have arrived at a composition which is a modification of a solution suggested by Bansi et a1 (2) , the main difference being that the concentration of essential amino acids in our solution is about twice that of Bansi et al., where- as the amount and type of non-essential nitrogen, i.e. 35 g glycine per litre, is the same in both solutions. Our solution has an E/T value (milligrams of the essen- tial amino acids per g of total nitrogen) of 2.6 ( 6 ) .

This paper reports the results on healthy volunteers; the results on clini- cal cases after surgery will be reported elsewhere. In the reported experiments the metabolic effect of the amino acid solution was compared with a commer- cial solution made by dialysis of digested casein having an E/T value of 3.0. In this solution about two thirds of the casein has been digested to individual amino acids, whereas the remainder con- sists of dialysable peptides ( 18).

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TABLE I. Composition of the solutions used for parentcral nitrogen nutrition.

According to analysis

According to Our Digested manufacturer* solution* casein

I-Threonine g/lit I-Valine g/lit I-Methionine g/lit 1 -1soleucine g/lit I-Leucine g/lit 1-Phenylalanine g/lit I-Tryptophane g/lit I-Lysine I-Histidine g/lit 1-Arginine g/lit Glycine g/lit Total nitrogen g/lit a-amino nitrogen g/lit Ammonia nitrogen mg/lit

Sorbitol g/lit

Calories per lit

PH

2.0 3.2 4.4 2.8 4.4 4.4 1 .o 3.2 2.2 4.5

34.0

50.0

2.0 3.3 4.5 2.9 4.4 4.4 1 .o 3.3 2.2 4.5

35.0 10.5 9.3 0.2-0.8

50.0

2 00

5.0

3.9 4.7 4.0 6.5

10.2 6.2 1 .o 6.6 2.3 2.6 2.0

14.3 8.6

15-85

-

-

5.0

* This solution (ASTRA 1738) has been made available by ASTRA, Sodetalje, Sweden.

Experimental Table I records the composition of the solution for parenteral amino acid nutrition used in all the experiments. One litre of the solution contains the eight essential amino acids in concentra- tions corresponding to about four times the quantities given by Rose (13) as the minimum requirements for man. The solution also contains the semi-essential amino acids, histidine and arginine, and - for provision of non-essential nitro- gen - 35 g glycine per litre. For increase of the caloric value 50 g sorbitol per litre was added, as suggested by Bansi et a1 ( 2 ) .

Table I also records the composition of the commercial solution of digested and dialysed casein used for comparison. The bulk concentration of a-amino groups is very similar in both solutions.

The experiments were carried out on nine reliable, adult healthy subjects, divided into four groups.

Group 1 (Fig. 1). Three women had nothing to eat for 15 consecutive days except the standard diet recorded in Table 11. Water ad libidem. This diet corresponded to 2.23 g nitrogen and 2240 calories per day. On the 8th and 9th day of the experiment one litre of the amino acid solution was given intra-

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TAELE 11. Hospital nitrogen-poor food, daily and 3, no food was given on the days of infusion. Instead she received, in addition ration to the amino acids, 500 ml of a com- mercial, nitrogen-free fat-emulsion and 1000 ml of a 20 per cent fructose solu-

White soft bread 22.2 1.47 0.33 White crisp bread 12.7 .60 o.20 tion intravenously, equivalent to 2000 Rice pancake 80.- 0.69 0.55 calories. On the 7th day she again ate

Weight g N % N g/day Type

Celery croquette 64.- 0.51 0.33 only the original diet. In this case no Mushroom sauce 96.- 0.53 0.51 comparison with the digested casein was

15.7 O.O1 made. The results from this subject are. Lingonberries Orange marmalade 34.1 0.03 0.01

recorded in Fig. 4. The other subject in Apple I00 0.04 0.04 Potatoes 75.- 0.33 0.25 this group discontinued the experiment

Sum 2.23

Calories 2240

venously over a period of 4 hours, and on the 12th and 13th day one litre of the digested casein solution. The experiment was concluded by two days (the 14th and 15th) on the standard diet only.

Group 2 (Fig. 2 ) . Exactly the same experiment as in group 1 was carried out on two men.

Group 3 (Fig. 3). The corresponding experiment as in group 1 was carried out on two women, except that the di- gested casein solution was given on the 8th and 9th day and the amino acid solution on the 12th and 13th day.

Group 4. Two 20 year old women re- ceived a diet similar to that used in the previous groups, but containing more calories and nitrogen (3.6 g per day) than the diet recorded in Table 11. The diet was given for 4 days only. One of the subjects had an infusion of 1 litre of the amino acid solution on the 5th and the 6th day of the experiment, but in contrast to the subjects in group 1, 2,

after the first day of infusion. The result turned out very similar to the other experiment in this group.

The intravenous infusions were given under constant supervision in the hos- pital. They were started about 8 oclock in the morning and lasted about 4 hours. Venous plasma samples were taken be- fore the start of the diet, immediately before, during, and immediately after the infusions.

All 24-hour urine samples were ana- lysed for total nitrogen (micro-Kjel- dahl), urea ( 3 ) , creatinine (9) , am- monia (3) a-amino nitrogen (15), and pH. Most of the mine samples were analysed for the individual amino acids by chromatography according to Moore and Stein. The faeces was analysed for total nitrogen only (micro-Kjeldahl). The plasma samples were analysed for ammonia only ( 3 ) .

Except for the time of infusion, the volunteers pursued their normal routinr during the period of the experiment. None of the subjects experienced any side-effects, except for the fact that they considered the food monotonous and that some of them felt hungry.

285

g N/day

+15 +1L- +13- +12- +11* +lo. +9.

+ 7- + 6. + 5 + A . + 3.

+a-

1 2 3 4 5 6 7 8 9 10 11 12 13 1L 15 Day t t t t

Am. acid Digcst.Cas. 10.29 1C3g N/day N/day

Unidentified-N gN/day +3- +2-

-1 -

a-amino-N

0 Cr-N

1= 0

3 2 0 1 :h 1 2 3 4

Urea-N

A;n.acib Digest.&. 10.2 9 1L.3 g N/day Nlday

Fig. 1. The effect on the nitrogen balance (left) and on the urine excretion of nitrogenous substances (right) by the amino acid solution and by the solution of digested and dialysed casein described in table I. During the entire 15 days of the experiment, including the days of infusion, the subjects had nothing to eat except the standard diet described in table I. O n the 8th and 9th day 1 litre of the amino acid solution was infused intravenously for a period of about 4 hours. O n the 12th and 13th day 1 litre of the solution of digested casein was administered in the same way. The diagram records the mean values from identical experiments on three healthy female volunteers, aged 31-48 years.

Results The metabolic results are reported only in the figures and only as mean values from each group (except for group 4) , since the results were surprisingly similar in all experiments in the individual groups. Only the daily nitrogen loss from the faeces varied considerably and without any correlation to the diet and the infusions. As this loss

was of little influence on the nitrogen balance, it is not recorded. The same experience has been reported previously (14). Since the amino acid pattern of the urine was not examined in all the cases, no mean values have been cal- culated. Instead the results from one of the subjects in group 1 are illustrated in Fig. 5. Also these patterns turned out to be unexpectedly similar in all experi-

286

+151 +14. +13. +12* +11. +lo. + 9. +8. + 7- + 6- + 5. +4. + 3.

m

1 2 3 4 5 6 7 8 9 10?l12131415Dav

Unidentlfffl-N g N/day

0 L=naddL 1 NH3-N

0 'r a- amino- N

21

0 l&

Cr -N

0 l-

Urea-N

5 4 3 2 t 1 1 O L

1 2 3 4 5 6 i 1 8 9 r t t t t

Am.=id Digest.cas 10.2 g 14.3 g N/day N/day

Fig. 2 . The same experiment as in Fig. 1 carried out on two healthy male volunteers, 34 and 44 years old.

ments subjected to chromatography. The mean values for plasma ammonia from all the subjects in groups 1, 2 and 3 are recorded in Table 111.

Discussion

The experiments in the groups 1 and 3 differed only with respect to the order in which the two test solutions (amino acid solution and casein hydrolysate) were given. The experiments in group 2 were identical with those in group 1, except for the fact that group 1 consisted of

M omen and group 2 of men. As expected, the results from all these groups were similar except for the magnitude of the nitrogen balance. During the days when only the standard food was given, this balance was more negative in the men than in the women, and during the days of infusion it was less positive. This is entirely in accordance with the fact that men have a greater requirement than women for nitrogen and calories (17) . For these reasons these three groups are discussed together. The fact that the 24

287

g N/day

+154 + 14. +13. + 12. +11. +lo. + 9. + 8- + 7. + 6. + 5- +4* +3.

rT-

g N/dry Unidentified-N

L d t 4 L m n 0 NH3-N

r 0 a-amino-N

Cr-N

l 1 0

8 7 6 5 I 3 2 1 0

-1 - 2 -3

1 2 3 4 5 6 7 8 9 1011 12131415Day 1 2 3 L 5 6 7 8 9 10ll1: r t t t t t 7

Urea-N

Irl 13 t 14 15 Day Digest.(%. Am. acM

14.3 g 10.2g Wday Wday

MgcstCas. Am.acM

N/day N/day u.3 g 10.2 g

Fig. 3. The corresponding experiment as in Fig. 1 carried out on two healthy female volunteers 32 and 33 years of age, differing from the experiments in Fig. 1 in that the order of infu- sions was reversed. The infusion of digested casein was given on the 8th and 9th day, and the amino acid solution on the 12th and 13th day.

hours creatinine excretion was remark- ably constant in each experiment is a check on the quantitative saving of the urine.

The standard food was prepared to give an insufficient supply of nitrogen, since it was expected that the nutritive effect of the solution on trial would be better evaluated by experiments on sub- jects in nitrogen deficit rather than in subjects with a positive nitrogen balance. Probably, a still more nitrogen-deficient diet would have been advantageous, but it was considered that this diet was the lower limit at which reliable subjects would volunteer. With respect to calories,

the nutritive value of the standard food was considered satisfactory, which is of importance for optimal utilisation of the nitrogen .

During the first days of the insufficient diet the nitrogen balance became con- siderably negative, with a tendency to approach a less negative but more stable level after a few days. This is in accord- ance with previous observations of the so called labile body protein first leaving the body during protein starva- tion, followed by a lower excretion of the endogenous nitrogen ( 1, 6 ) . The results would probably have been more clearcut if the subjects had been main-

288

TABLE 111. Plasma ammonia in 7 healthy subjects before and after parenteral nitrogen administration, ng/ml

~ ~~ ~~~

Amino acid. sol. Digested casein sol.

1 st 2nd 1st 2nd

inf. inf. inf. inf.

Before 0.69 0.82 0.65 0.80 After 0.84 1.05 1.15 1.33 Difference* 0.15 0.23 0.50 0.53

* Both the differences 0.50-0.15 and 0.53- 0.23 are statistically highly significant (p< 0.001 ).

tained on the standard diet for a longer period before the infusions ( 7 ) .

In all the experiments the nitrogen balance became positive when one litre of the nutrition solution was ad- ded by infusion to the constant oral supply of 2.2 g nitrogen per day in the standard diet. Invariably, the nitrogen balance was more positive on the first day of parenteral nutrition than on the second day. The most probable explana- tion of this.observation is that the main part of the nitrogen deficit is filled up already by the first days infusion.

In all three groups the nitrogen bal- ance became more positive when the digested casein was given, than after the infusion of the amino acid solution, even if counted in per cent of the infused amount of nitrogen. However, the dif- ference between the two solutions, with respect to the nitrogen balance, was not great enough to be significant for the nutrition.

As will he demonstrated in another paper, soon to be published, the dif-

D N n o i v e d l N b r l a r a

g N / d y +11.

Am r i d

N / d y 10.2 g

a-amino-N

0 L d L Cr-N A-

Urea-N

Am. s i d m.2 9 N/day

Fig. 4. The effect of intravenous infusion of 1 litre of the amino acid solution on the nitrogen balance and excretion in one healthy, 20 year old female volunteer who had nothing to eat except a standard food containing 3.8 g of nitrogen a day for 4 days prior to the infusion, but (in contrast to the experiments in Fig. 1-3) nothing to eat during the day of infusion. In this experiment no digested casein was given.

ference was the opposite after surgical operations. In these cases the nitrogen balance was more advantageous to the patients when the amino acid solution was given. This discrepancy indicates that the amino acid requirements may be different in health and after a major trauma, e.g. operation. In the normal cases the infusion had to fill up a deficit of mainly labile body protein, whereas the surgical cases must be losing con- siderable quantities of endogenous ceil protein.

The results from this investigation dif- fer from those of Bansi et a1 (2 ) in that a positive nitrogen balance was invariab-

289

3.0-

2.5.

2.0.

1.5.

1.0.

0.5.

0- THREON. GLYC. .. I I. N.

8.2

J. PHE -AL. TR'iPT. L I

;. H k .

0 1-Amino acid m synth.sd. fl 1-Amino acid in dgest.sd. I Amino acid excreted Fig. 5. The quantities of administered amino acids and the Moore and Stein pattern of amino acids in the urine from the 2 days of amino acid infusion and the 2 days of infusion of digest- ed and dialysed casein in one of the female volunteers recorded in Fig. 1 .

ly found when the amino acid solution was administered, whereas Bansi et a1 always observed a negative balance with their solution. There were three main differences between the experiments of Bansi et a1 and those presented here: 1 ) The amino acid solution of Bansi et a1 had the same source and amount of non- essential nitrogen as ours, i.e. 35 g of glycine per litre, but only half the con- centration of the essential amino acids. This may be one reason for a less complete accumulation of nitrogen. 2 ) The subjects in the Bansi experiments were patients with gastric ulcers or gas- tritis, who may have had a more labile nitrogen balance than our healthy vol-

unteers. 3) During the days of infusion Bansi et a1 did not give their subjects any food, whereas our volunteers ate the same standard diet as during the days preceding the infusions. Consequently, it could not be ruled out that the essential amino acids provided with the food pro- tein may have rendered the nitrogen balance positive in our experiments. In order to examine the influence of the food on the results, the group 4 experi- ments were carried out. Even if no food was given, our solution turned the neg- ative nitrogen balance into a positive one, although less positive than in the other experiments (Fig. 4).

The pattern of nitrogen excretion in

290

the urine was almost identical in groups 1, 2 and 3. Whether the amino acids or the digested casein was given, the urea output increased by 3 to 4 g of nitrogen on the days of infusion. Without NI5 studies it is impossible to decide which amino acid it was that provided amino groups for this extra urea. According to the results of Sprinson and Rittenberg (16)) it would seem probable that the main source was the high proportion of glycine in the amino acid solution. How- ever, this explanation cannot be valid for the digested casein, since the same urea increase was observed when the casein solution, with its low glycine con- tent, was given.

The changes in urea output were al- most a mirror image of the correspond- ing changes of the nitrogen balance. The balance was always more positive on the first day of infusion than on the second day. In contrast to this, the wasting of urea was always higher on the second day of infusion. Thus, when the amino acid deficit starts being saturated, the excess amino acids are wasted as urea.

The excretion of a-amino nitrogen was always increased by about 1 g per clay, whichever solution was given. This is in accordance with the augmented amino acid excretion demonstrated in Fig. 5.

Also the ammonia excretion was a little higher during the days of infusion. The most probable reason is that the urine became a little more acid during the infusions. The increase in the ex- cretion of amino acids and ammonia was of little influence on the nitrogen balance.

The quantity of unidentified nitro-

gen should always be slightly positive, since it represents the difference between total urine nitrogen, determined by the Kjeldahl method, and the sum of the nitrogen recovered as urea, creatinine, u-amino groups, and ammonia. It thus includes nitrogen from uric acid, crea- tine, purines etc. in the urine. The fact that this quantity was very low during the days on only the standard food may be considered as a check on the respec- tive analyses.

It should be observed that the %n- identified urine nitrogen was always increased when the digested casein was given, but not after the amino acid solution. As will be demonstrated in another paper, this extra unidentified nitrogen increased to a greater extent when the hydrolysate was administered to recently operated patients. It has been demonstrated that this nitrogen consists of peptides, since hydrolysis of this frac- tion deliberates a corresponding amount of a-amino nitrogen (11). I t has been pretended (10) that the digested casein solution should not exert any disagree- able side-effects. However, some of our operated patients (but none of the healthy volunteers) felt nauseated or vomited on receiving 1 litre of the digest- ed casein, whereas the amino acid solu- tion did not cause any discomfort. It seems probable that the peptides were responsible for these side-effects.

The Moore and Stein analyses of the amino acid pattern of the urine during the days of infusion revealed that about 10 per cent of the administered glycine was wasted, irrespective whether the amino acids, including 35 g of glycine, or the hydrolysate with 2 g of this com-

29 1

pound were given. Of the other amino acids, only histidine appeared in quanti- ties of any significance. The histidine excretion had been previously observed by Bansi and coworkers (2) who related it to the limited capacity of the renal tubules to reabsorb this amino acid. The fact that also threonine had a slight tendency to be increased in the urine when the amino acids were infused, may depend on competition by the glycine for tubular reabsorption in the kidneys ( 8 ) .

As demonstrated in Table 111, the blood ammonia concentration increased slightly by infusion of both the solutions, the increase during administration of the digested casein being significantly higher than when the amino acid solution was given. However, also during infusion of the hydrolysate the ammonia concentra- tion was well below the level at which subjective symptoms of ammonia in- toxication appear. Thus, the disagree- able symptoms sometimes appearing during such an infusion can scarcely be due to an increase of the blood am- monia. The observation that an amino acid infusion increased the blood am- monia content less than an infusion of casein hydrolysate may depend on the higher amount of arginine in the former solution. I t has been found (12) that arginine may capture ammonia, bringing it into the Krebs-Henseleit cycle. This explanation may be valid, even if ar- ginine has no effect on the increase of ammonia in cases of liver insufficiency

In contrast to published reports that glycine may exert toxic influences (12), we have not observed any side-effects from the amino acid solution. On the

( 5 ) .

contrary, we can confirm previous ob- servations (2 , 7) that glycine is a pos- sible way to supply non-essential ni- trogen, provided that the essential amino acids and arginine are administered in sufficient quantities.

In papers, soon to be published, the effect of the amino acid solution on operated, surgical cases will be reported. We will also report the effect of other solutions, including ammonium salts, on the nitrogen balance in human subjects.

Summary

A solution containing the essential amino acids and histidine and arginine, and - as a source of non-essential nitrogen - a sufficient amount of glycine is sug- gested for intravenous nitrogen nutrition in man. The nutritional effect of this solution was tested on 9 healthy volun- teers. In all the subjects a negative nitrogen balance, due to nitrogen star- vation, turned positive when 1 litre of the solution was given per day. No un- pleasant side-effects were observed.

References

ALBANESE, A. : Criteria of protein nutrition. Protein and Amino Acid Nutrition, Ac. Press, 297, 1959. BANSI, H. W., JURGENS, P., MULLER, G . & ROSTIN, M.: Der Stoffwrchsel bei intra- venoser Applikation von Nihrliisungen, insbesondere synthetisch zusammengestell- ten Aminosaurelosungen. Klin. Woch. 332, 1964. CHANEY, A. & MARBACH, E.: Modified reagents for determination of urea and ammonia. Clin. Chem. 8: 130, 1962.

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4. ELMAN, R. & WEINER, D. 0.: Intravenous alimentation (with special reference to protein (amino acid) metabolism). J. Amer. Med. Ass. 112: 796, 1939.

5. FAHEY, J., NATHAN, D. & RAIRIGH, D.: Effect of L-arginine on elevated blood ammonia levels in man. Amer. J. Med. 23: 860, 1957.

6. FAO/WHO Expert Group: Protein Require- ments. World Health Organization Tech- nical Report Series 301, 1965.

7. FROST, D.: Methods of measuring the nutritive value of proteins, protein hydro- lysates and amino acid mixtures. The repletion method. Protein and Amino acid Nutrition, Ac. Press, 225, 1959.

8. HANDLER, P., KAMIN, H. & HARRIS, J.: The metabolism of parenterally administe- red amino acids. J. biol. Chem. 179: 283, 1949.

9. HAUGEN, H. N.: The determination of endogenous creatinine in plasma and urine. Scand. J. clin. Lab. Invest. 5: 48, 1953.

10. LIDSTROM, F.: Clinical studies during parenteral nutrition with a dialyzed amino acid preparation (Aminosol). Acta Chir. Scand. Suppl. 186:37, 1954.

11. LIDSTROM, F. & WRETLIND, A,: Effect of dialyzed hydrolysate. Scand. J. Clin. Lab. Invest. 4: 167, 1952.

12. NAJARIAN, J. & HARPER, H.: Comparative effect of arginine and monosodium gluta- mate on blood ammonia. Proc. SOC. exp. Biol. Med. 92: 560, 1956.

13. ROSE, W. C. : Amino acid requirements of man. Fed. Proc. 8: 546, 1949.

14. SNYDERMAN, SELMA, HOLT, E., DANCIS, J., ROITMAN, ELLEN, BOYER, AUDREY & BALIS, E.: Unessential nitrogen: a limiting factor for human growth. J. Nutrition 78: 57, 1962.

15. SOBEL, C., HENRY, R., CHIAMORI, N. & SEGALOVE, M.: Determination of a- amino acid nitrogen in urine. Proc. SOC. exp. Biol. Med. 95: 808, 1957.

16. SPRINSON, D. & RITTENBERG, D.: The rate of utilization of ammonia for protein synthesis. J. biol. Chem. 180: 707, 1949.

17. SWENDSEID, M., HARRIS, C. & TUTTLE, S.: An evaluation of the F A 0 amino acid reference pattern in human nutrition. 11. Studies with young women. J. Nutrition 77: 391, 1962.

18. WRETLIND, A,: The amino acid content of a dialyzed enzymatic casein hydrolysate. Acta Physiol. Scand. 27: 189, 1952.

Reprints from professor Bertil Josephson, Clinical Central Laboratory, St Eriks Hospital, Stock- holm.

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