3. Results

Table 1 shows the comparison of mean values for the clinical and biochemical characteristics (blood and urine) in the diabetic and control groups. Fasting glycaemia, Hb1Ac, and GFR were significantly higher (p < 0.05) within the diabetic group compared to the control group. There were not any significant differences in age, BMI Z-score, systolic and diastolic blood pressure, and urinary albumin excretion between both groups.

There was no correlation between glomerular filtration and Hb1Ac or urinary albumin excretion, not between blood pressure (systolic and diastolic blood pressure) and glomerular filtration or Hb1Ac. There was a positive correlation (p < 0.05) between diastolic blood pressure and the evolution of the disease (years) (r = 0.515).

Table 2 exposes and compares the mean values of amino acid plasma concentrations for the samples of the diabetic and control groups. Plasma concentrations of ARG, GLN, ILE, PHE, THR, TYR, VAL, and TAU were significantly higher (p < 0.05) within the diabetic group with respect to the control group.

Table 3 depicts and compares the mean values of plasma concentrations of different amino acids groups analyzed in the diabetic and control group. The plasma levels of total amino acids as well as branched-chain, glucogenic, and ketogenic amino acids were significantly higher (p < 0.05) in the diabetic group with respect to the control group.

There was no correlation between the single amino acid (or amino acid groups) plasma levels and the evolution of the disease (years) or Hb1Ac. There was a negative correlation (p < 0.05) among insulin dosage and amino acids THR (r = 0.404), MET (r = 0.513), PHE (r = 0.456), SER (r = 0.442), CYS (r = 0.390), GLY (r = 0.451), and TAU (r = 0.479), as well as a positive correlation (p < 0.05) among glycaemia and amino acids VAL (r = 0.545) and LEU

Table 2. Plasma concentrations of amino acids (nmol/ml) in the diabetic and control groups (M SD).

ALA: alanine, ARG: arginine, ASP: aspartic acid, CYS: cysteine, GLN: glutamine, GLU: glutamic acid, GLY: glycine, HIS: histidine, ILE: isolecucine, LEU: leucine, LYS: lisine, MET: methionine, PHE: phenylalanine, SER serine, THR: threonine,

Control group (n = 48)

Amino Acid Plasma Concentrations and Urinary Excretion in Young Diabetics

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134.84 36.67 22.62 6.94 1.34 2.18 31.56 10.90 187.84 56.83 20.70 10.22 35.34 10.23 147.09 53.89 66.54 15.27 68.65 14.65 57.15 28.61 29.18 13.05 65.64 16.45 66.04 22.04 57.90 18.07 38.25 12.47 148.91 35.31 75.66 37.01

Control group (n = 48)

p-Values

113

p-Values

n.s. <0.01 n.s. n.s. <0.01 n.s. n.s. n.s. <0.001 n.s. n.s. n.s. <0.01 n.s. <0.05 <0.05 <0.01 <0.05

Table 4 displays and compares the mean values of urinary concentrations of different amino acids quantified in the diabetic and control group. The urinary level of amino acids, except for ASP, ILE, and PHE, was significantly lower (p < 0.05) in the diabetic group with respect to the

Table 5 outlines and compares the mean values of urinary levels of the different amino acids groups in the diabetic and control group. Total as well as branched-chain, glucogenic and ketogenic amino acid urinary levels were significantly lower (p < 0.05) in the diabetic group compared to the control group. The mean values of the glucogenic/total amino acid ratio were significantly lower (p < 0.05) in the diabetic group with respect to the control group. There were no significant differences in the ketogenic/total amino acid ratio between both groups.

Total 1383.70 353.67 1198.46 261.16 <0.05 Branched-chain 347.65 58.76 285.20 45.20 <0.01 Glucogenic 1252.74 236.82 1053.69 211.19 <0.001 Ketogenic 441.62 57.09 354.13 53.45 <0.05

Table 3. Plasma concentrations of amino acids (nmol/ml) in the diabetic and control groups (M SD).

(r = 0.648).

TYR tyrosine, VAL: valine, TAU: taurine.

Amino acid groups Diabetic group

(n = 49)

ALA ARG ASP CYS GLN GLU GLY HIS ILE LEU LYS MET PHE SER THR TYR VAL TAU

Amino acids Diabetic group

(n = 49)

144.83 36.32 49.01 16.78 0.33 0.95 34.77 11.61 243.23 59.42 18.28 9.69 46.53 22.3 133.62 43.59 90.83 19.37 74.74 17.37 60.70 27.32 31.57 10.68 81.84 19.54 53.93 26.03 73.26 27.90 60.25 27.18 190.46 48.01 99.69 36.82

control group.


GFR: glomerular filtration rate, UAE: urinary albumin excretion.

Table 1. Clinical and biochemical characteristics of the diabetic and control groups (M SD).

Amino Acid Plasma Concentrations and Urinary Excretion in Young Diabetics http://dx.doi.org/10.5772/intechopen.72080 113


3. Results

112 Diabetes and Its Complications

Table 1 shows the comparison of mean values for the clinical and biochemical characteristics (blood and urine) in the diabetic and control groups. Fasting glycaemia, Hb1Ac, and GFR were significantly higher (p < 0.05) within the diabetic group compared to the control group. There were not any significant differences in age, BMI Z-score, systolic and diastolic blood pressure,

There was no correlation between glomerular filtration and Hb1Ac or urinary albumin excretion, not between blood pressure (systolic and diastolic blood pressure) and glomerular filtration or Hb1Ac. There was a positive correlation (p < 0.05) between diastolic blood pressure and

Table 2 exposes and compares the mean values of amino acid plasma concentrations for the samples of the diabetic and control groups. Plasma concentrations of ARG, GLN, ILE, PHE, THR, TYR, VAL, and TAU were significantly higher (p < 0.05) within the diabetic group with

Table 3 depicts and compares the mean values of plasma concentrations of different amino acids groups analyzed in the diabetic and control group. The plasma levels of total amino acids as well as branched-chain, glucogenic, and ketogenic amino acids were significantly higher

There was no correlation between the single amino acid (or amino acid groups) plasma levels and the evolution of the disease (years) or Hb1Ac. There was a negative correlation (p < 0.05) among insulin dosage and amino acids THR (r = 0.404), MET (r = 0.513), PHE (r = 0.456), SER (r = 0.442), CYS (r = 0.390), GLY (r = 0.451), and TAU (r = 0.479), as well as a

Age (years) 11.82 1.78 12.05 1.93 n.s. BMI Z-score 0.05 0.67 0.01 0.55 n.s. Systolic BP 93.15 8.85 89.0 8.95 n.s. Diastolic BP 55.0 7.26 52.2 8.36 n.s. Evolution (years) 5.79 2.67 — — Insulin (UI/kg/d) 0.82 0.26 — — Glucose (mg(dl)) 198.8 55.5 89.57 10.2 <0.01 Hb1Ac (%) 7.7 1.68 4.56 0.7 <0.05

UAE (ug/min) 3.77 1.8 3.42 1.89 n.s.

Table 1. Clinical and biochemical characteristics of the diabetic and control groups (M SD).

) 135.65 34.3 114.16 9.13 <0.05

Control group (n = 48)

p-Values

and urinary albumin excretion between both groups.

(p < 0.05) in the diabetic group with respect to the control group.

(n = 49)

the evolution of the disease (years) (r = 0.515).

Items Diabetic group

GFR: glomerular filtration rate, UAE: urinary albumin excretion.

respect to the control group.

GFR (ml/min/1.73 m<sup>2</sup>

ALA: alanine, ARG: arginine, ASP: aspartic acid, CYS: cysteine, GLN: glutamine, GLU: glutamic acid, GLY: glycine, HIS: histidine, ILE: isolecucine, LEU: leucine, LYS: lisine, MET: methionine, PHE: phenylalanine, SER serine, THR: threonine, TYR tyrosine, VAL: valine, TAU: taurine.

Table 2. Plasma concentrations of amino acids (nmol/ml) in the diabetic and control groups (M SD).

positive correlation (p < 0.05) among glycaemia and amino acids VAL (r = 0.545) and LEU (r = 0.648).

Table 4 displays and compares the mean values of urinary concentrations of different amino acids quantified in the diabetic and control group. The urinary level of amino acids, except for ASP, ILE, and PHE, was significantly lower (p < 0.05) in the diabetic group with respect to the control group.

Table 5 outlines and compares the mean values of urinary levels of the different amino acids groups in the diabetic and control group. Total as well as branched-chain, glucogenic and ketogenic amino acid urinary levels were significantly lower (p < 0.05) in the diabetic group compared to the control group. The mean values of the glucogenic/total amino acid ratio were significantly lower (p < 0.05) in the diabetic group with respect to the control group. There were no significant differences in the ketogenic/total amino acid ratio between both groups.


Table 3. Plasma concentrations of amino acids (nmol/ml) in the diabetic and control groups (M SD).


pathway—through the glyconeogenesis [1, 2, 13]. This might explain the differences found in the amino acid plasma levels within the diabetic and control group that, in general, would indicate that there is an increase in the bioavailability of glycogenic substrates in diabetic

Amino Acid Plasma Concentrations and Urinary Excretion in Young Diabetics

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115

Insulin leads to a decrease in amino acid plasma levels through the stimulus of protein synthesis and the inhibition of proteolysis [2, 14]; this would largely explain the negative correlation found between insulin dosage and plasma level of the different amino acids. Therefore, the significantly high plasma concentrations of the different amino acids–particularly glucogenic amino acids–in the diabetic group with respect to the control group (probably as a consequence of the insulinopenia that characterizes diabetes), could be useful as plasma

An increase in postprandial branched-chain amino acid (valine, leucine, and isoleucine) plasma concentrations has been described in diabetic patients, in relation to the metabolic control of the disease [4, 15]. This is probably due to a deficient peripheral metabolism of these amino acids (they undergo basically muscle metabolism). Since it has not been possible to prove an increase in the release of branched-chain amino acids from muscle and/or liver in diabetic patients during fasting [1, 2], and being conscious that biological effects of insulin are deficient in diabetes, it can be assumed that the increased branchedchain amino acid plasma levels in the diabetic group in comparison to the control group would be due to a low stimulation (by insulin) in amino acid transportation inside the cell. Even though no correlation has been found between branched-chain amino acids and metabolic control or time of evolution of diabetes, a positive correlation has been detected between valine and leucine and fasting glycaemia, a fact that would support the hypothesis of a more intense relationship among basal plasma concentration of these amino acids and single determination glycaemia rather than with mediumterm metabolic

Even though amino acids are appropriate substrates for hepatic and/or renal synthesis of glucose (gluconeogenesis), glutamine and especially alanine are the most important glucogenic amino acids in quantitative terms [16]. However, while glutamine plasma levels in basal conditions were significantly higher in the diabetic group in comparison to control group, alanine plasma

During fasting, alanine release does not exclusively correspond to a mechanism of proteolysis and posits muscle synthesis of new molecules of alanine from the glucose captured by the muscle or glucose alanine cycle [2, 16]. Nevertheless, since glucose uptake by the muscle is lowered due to insulinopenia in the diabetic patient, the conversion of glucose into alanine would be decreased, and consequently, this would explain why alanine plasma levels in the

Amino acid metabolism in insulin-dependent diabetes mellitus appears to be intrinsically disrupted, since insulin deficiency and to a great extent, the effects of the counter regulatory hormones imply an increase in hepatic gluconeogenesis and muscle proteolysis, as well as a deficient peripheral use and/or disturbance in hepatic amino acid metabolism; this would result in a plasma profile characterized by an increase of total amino acids, at the expense

patients, even in basal conditions.

markers of a deficient metabolic control.

levels did not differ in those groups.

diabetic group do not differ from the control group.

control.

ALA: alanine, ARG: arginine, ASP: aspartic acid, CYS: cysteine, GLN: glutamine, GLU: glutamic acid, GLY: glycine, HIS: histidine, ILE: isolecucine, LEU: leucine, LYS: lisine, MET: methionine, PHE: phenylalanine, SER serine, THR: threonine, TYR tyrosine, VAL: valine, TAU: taurine.

Table 4. Urinary levels of amino acids (μmol/m<sup>2</sup> ) in the diabetic and control groups (M SD).


Table 5. Urinary level of amino acid groups in the diabetic and control groups (M SD).

There was no correlation between the levels of each particular amino acid and/or group of amino acids in urine and the time of evolution, Hb1Ac, urinary albumin excretion, GFR, and blood pressure (systolic and diastolic blood pressure).

#### 4. Discussion

#### 4.1. Plasma concentrations

In diabetes mellitus, the deficiency in insulin, and in large part, the effects of the counter regulatory hormones would stimulate the synthesis of glucose—other than the glycogenolysis pathway—through the glyconeogenesis [1, 2, 13]. This might explain the differences found in the amino acid plasma levels within the diabetic and control group that, in general, would indicate that there is an increase in the bioavailability of glycogenic substrates in diabetic patients, even in basal conditions.

Insulin leads to a decrease in amino acid plasma levels through the stimulus of protein synthesis and the inhibition of proteolysis [2, 14]; this would largely explain the negative correlation found between insulin dosage and plasma level of the different amino acids. Therefore, the significantly high plasma concentrations of the different amino acids–particularly glucogenic amino acids–in the diabetic group with respect to the control group (probably as a consequence of the insulinopenia that characterizes diabetes), could be useful as plasma markers of a deficient metabolic control.

An increase in postprandial branched-chain amino acid (valine, leucine, and isoleucine) plasma concentrations has been described in diabetic patients, in relation to the metabolic control of the disease [4, 15]. This is probably due to a deficient peripheral metabolism of these amino acids (they undergo basically muscle metabolism). Since it has not been possible to prove an increase in the release of branched-chain amino acids from muscle and/or liver in diabetic patients during fasting [1, 2], and being conscious that biological effects of insulin are deficient in diabetes, it can be assumed that the increased branchedchain amino acid plasma levels in the diabetic group in comparison to the control group would be due to a low stimulation (by insulin) in amino acid transportation inside the cell. Even though no correlation has been found between branched-chain amino acids and metabolic control or time of evolution of diabetes, a positive correlation has been detected between valine and leucine and fasting glycaemia, a fact that would support the hypothesis of a more intense relationship among basal plasma concentration of these amino acids and single determination glycaemia rather than with mediumterm metabolic control.

Even though amino acids are appropriate substrates for hepatic and/or renal synthesis of glucose (gluconeogenesis), glutamine and especially alanine are the most important glucogenic amino acids in quantitative terms [16]. However, while glutamine plasma levels in basal conditions were significantly higher in the diabetic group in comparison to control group, alanine plasma levels did not differ in those groups.

During fasting, alanine release does not exclusively correspond to a mechanism of proteolysis and posits muscle synthesis of new molecules of alanine from the glucose captured by the muscle or glucose alanine cycle [2, 16]. Nevertheless, since glucose uptake by the muscle is lowered due to insulinopenia in the diabetic patient, the conversion of glucose into alanine would be decreased, and consequently, this would explain why alanine plasma levels in the diabetic group do not differ from the control group.

There was no correlation between the levels of each particular amino acid and/or group of amino acids in urine and the time of evolution, Hb1Ac, urinary albumin excretion, GFR, and

ALA: alanine, ARG: arginine, ASP: aspartic acid, CYS: cysteine, GLN: glutamine, GLU: glutamic acid, GLY: glycine, HIS: histidine, ILE: isolecucine, LEU: leucine, LYS: lisine, MET: methionine, PHE: phenylalanine, SER serine, THR: threonine,

Total 754.94 427.16 1868.42 662.36 <0.05 Branched-chain 54.58 26.51 101.13 36.76 <0.05 Glucogenic 252.51 178.75 943.79 370.50 <0.05 Ketogenic 236.40 121.16 530.69 215.78 <0.05 Ratio G/T 0.34 0.09 0.50 0.07 <0.05 Ratio K/T 0.33 0.16 0.28 0.12 n.s.

) in the diabetic and control groups (M SD).

Control group (n = 48)

In diabetes mellitus, the deficiency in insulin, and in large part, the effects of the counter regulatory hormones would stimulate the synthesis of glucose—other than the glycogenolysis

blood pressure (systolic and diastolic blood pressure).

Amino acids Diabetic group

TYR tyrosine, VAL: valine, TAU: taurine.

G/T: glucogenic/total, K/T: ketogenic/total.

Table 4. Urinary levels of amino acids (μmol/m<sup>2</sup>

Amino acid groups Diabetic group

(n = 49)

Table 5. Urinary level of amino acid groups in the diabetic and control groups (M SD).

ALA ARG ASP CYS GLN GLU GLY HIS ILE LEU LYS MET PHE SER THR TYR VAL TAU

114 Diabetes and Its Complications

(n = 49)

53.97 36.68 2.45 1.91 4.98 3.23 19.25 11.07 7.84 4.52 16.62 9.61 23.00 15.05 74.12 48.50 18.98 10.31 12.46 8.17 223.65 150.75 19.11 8.01 40.64 22.58 10.64 7.64 14.47 10.96 26.65 16.28 25.61 11.94 115.17 56.70

Control group (n = 48)

118.07 45.24 4.59 2.97 8.84 4.22 61.91 29.17 95.02 32.18 35.83 15.77 192.41 121.59 233.95 89.36 29.83 18.42 22.16 9.13 525.32 196.31 86.04 56.06 51.67 13.71 25.40 13.65 63.29 19.36 79.08 21.15 45.11 13.67 172.31 107.12 p-Values

<0.001 <0.05 n.s. <0.05 <0.001 <0.05 <0.001 <0.01 n.s. <0.05 <0.05 <0.05 n.s. <0.01 <0.05 <0.001 <0.05 <0.01

p-Values

4. Discussion

4.1. Plasma concentrations

Amino acid metabolism in insulin-dependent diabetes mellitus appears to be intrinsically disrupted, since insulin deficiency and to a great extent, the effects of the counter regulatory hormones imply an increase in hepatic gluconeogenesis and muscle proteolysis, as well as a deficient peripheral use and/or disturbance in hepatic amino acid metabolism; this would result in a plasma profile characterized by an increase of total amino acids, at the expense mainly of branched-chain and glucogenic amino acids. In this way, the study of the amino acid plasma profile in diabetic patients would be worthwhile, since it would reflect disturbances in protein and/or amino acid metabolism and consequently, in metabolic control of the disease.

Author details

Spain

References

Manuel Moya-Benavent<sup>3</sup>

Teodoro Durá-Travé1,2\*, Fidel Gallinas-Victoriano2

England: Wiley; 2004. pp. 319-334

Clinical Practice. 1991;12:91-97

Rinsho. 1992;50:1631-1636

1572-1579

patients. Diabetes Research 1989;12:57-62

type 1 diabetes. Diabetes. 2002;51:1580-1587

Journal of Nephrology. 1996;16:300-303

\*Address all correspondence to: tduratra@cfnavarra.es

1 Department of Pediatrics, School of Medicine, University of Navarre, Pamplona, Spain

3 Department of Pediatrics, School of Medicine, University of "Miguel Hernández", Elche,

[1] Kimball SR, Flaim KE, Peavy DE, Jefferson LS. Protein metabolism. In: Rifkin H, Porte D, editors. Diabetes Mellitus. Theory and practice. New York: Elsevier SP; 1990. pp: 41-50 [2] Liu Z, Barret EJ. Protein metabolism in health and diabetes. In: Defronzo RA, Ferrannini E, Keen H, Zimmet P, editors. International Textbook of Diabetes Mellitus. Chichester,

[3] Trevisan R, Marescotti C, Avogaro A, Tessari P, del Prato S, Tiengo A. Effects of different insulin administrations on plasma amino acid profile in insulin-dependent diabetic

[4] Szabó A, Kenesei E, Körner A, Miltényi M, Szücs L, Nagy I. Changes in plasma and urinary amino acid levels during diabetic ketoacidosis in children. Diabetes Research and

[5] Ohtsuka Y, Agishi Y. Abnormal amino acid metabolism in diabetes mellitus. Nihon

[6] Adeva MM, Calviño J, Souto G, Donapetry C. Insulin resistance and the metabolism of

[7] Drummond K, Mauer M, International Diabetic Nephropathy Study Group. The early natural history of nephropathy in type 1 diabetes: II. Early renal structural changes in

[8] Mauer M, Drummond K. The early natural history of nephropathy in type 1 diabetes: I. Study design and baseline characteristics of the study participants. Diabetes. 2002;51:

[9] Hsiao PH, Tsai WS, Tsai WY, Lee JS, Tsau YK, Chen CH. Urinary N-acetyl-beta-Dglucosaminidase activity in children with insulin-dependent diabetes mellitus. American

branched-chain amino acids in humans. Amino Acids. 2012;43:171-181

2 Department of Pediatrics, Navarre Hospital Complex, Pamplona, Spain

, Ernesto Cortes-Castell<sup>3</sup> and

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#### 4.2. Urinary excretion

Diabetic nephropathy is preceded by a window period, which might show different renal functional and/or structural disturbances, even in the early stages of the disease [7, 8]. In fact, the results obtained, in line with other researchers [17, 18], reveal significantly higher glomerular filtration in the diabetic group in comparison to the control group, and especially in those patients with a shorter period of disease and regardless of metabolic control of the disease. In addition, even when the whole diabetic group had normal blood pressure measurements, the existing correlation between diastolic blood pressure and the time of evolution of the disease suggests a situation of window period in diabetic nephropathy in this group of young diabetics, and highlights the importance of periodic blood pressure measurements in diabetics from the early stages of the disease. This allows for the beginning of a dietary and/or medical treatment earlier than was recommended until now [19]. However, it can be concluded that the structural integrity of the glomerulus in these diabetic patients would be relatively well preserved, since the urinary excretion of albumin was similar in both groups.

On another note, several researchers have noted a higher beta 2 microglobulin and lysosomal enzyme urinary excretion in diabetic patients in the absence of microalbuminuria, as a sign of functional disorder in the proximal tubule with no glomerular lesion, from the early stages of the disease [10, 11]. In this context, the study of amino acid urinary excretion in the diabetic could be of great interest, since different mechanisms of specific tubular reabsorption for different amino acids have been described on an experimental basis [20]. Hence, any tubular malfunction might condition significant qualitative and/or quantitative aminoaciduria, and therefore, it could have a potential clinical application in the early detection of tubular lesion and/or silent diabetic nephropathy.

All the same, and according to the results obtained, urinary excretion of each single amino acid (except for isoleucine, aspartic acid, and taurine), as well as each amino acid groups analyzed were significantly lower in the diabetic group with respect to the control group. This may seem paradoxical; however, the difference observed in the relation glucogenic and total amino acid (G/T ratio) between both groups reveals that the lower amino acid urinary excretion in the diabetic would greatly be at the expense of glucogenic amino acids, probably because the glomerular filtration is also lower, as a consequence of a greater organic use of these amino acids in the endogenous synthesis of glucose. No correlation has been found between aminoaciduria and time of evolution, glomerular filtration, blood pressure, and metabolic control.

In sum, the study of amino acid urinary excretion in the young diabetic might have interest not only in the context of diabetic nephropathy, but also in the revealing of partial aspects of amino acid metabolism, and probably, in the metabolic control of the disease.
