2. Material and methods

#### 2.1. Participants

A clinical assessment and metabolic study was accomplished in 49 children diagnosed with insulin-dependent diabetes mellitus, aged 8.6–14.3 years, following conventional insulin therapy, and a group of 48 healthy children (control group) aged 7.4–14.8 years.

#### 2.2. Clinical assessment

Information recorded from every patient/participant included age, weight and height, BMI, time and progress of the disease, and dosage of subcutaneous insulin.

Weight and height measurements were made in underwear while being barefoot. Weight was measured using the Año-Sayol scale (reading interval 0–120 kg and a precision of 100 g), and height was measured using the Holtain wall stadiometer (reading interval 60–210 cm, precision 0.1 cm). The Z-score values for the BMI were calculated using the epidemiologic data contained within the program Aplicación Nutricional, from the Spanish Society of pediatric gastroenterology, hepatology, and nutrition (Sociedad Española de Gastroenterología, Hepatología y Nutrición Pediátrica, available at http://www.gastroinf.es/nutritional/). The graphics from Ferrández et al. (Centro Andrea Prader, Zaragoza) (2002) were used as reference charts.

Blood pressure (BP) was measured in the right arm with the patient in the supine position using Visomat comfort 20/40 (Roche Diagnostics Inc.) digital blood pressure monitor, recording the lowest of three measurements.

#### 2.3. Biochemical analysis

It has been experimentally proven that insulin deficiency involves a series of ultrastructural and/or functional changes at an intracellular level, within muscle as well as liver, which substantially inhibit protein synthesis and stimulate protein degradation. Therefore, being amino acidosis, the structural elements of proteins and its metabolism could be altered in diabetes mellitus [1, 2]. In fact, significant changes in amino acid plasma levels and urinary excretion have been described in diabetic ketoacidosis, as well as anomalies in postprandial plasma profile of such amino acids in diabetic patients, whose values will not even return to

Diabetic nephropathy is one of the most frequent and severe late complications in infantjuvenile diabetes; its functional and structural pathology seems to be shaped from the early stages of the disease. Persistent microalbuminuria is a functional disruption that occurs in the emerging phases of diabetic nephropathy, whose early detection and monitoring is quite

An increased urinary excretion of low molecular weight proteins and lysosomal enzymes has been confirmed in diabetic patients in the absence of microalbuminuria, as a result of a disorder in renal tubular reabsorption; its significance in natural history of diabetic nephropathy would be interpreted as early markers of renal injury [9–11]. On the other hand, barely 2– 3% of the total amount of amino acidosis filtered by the glomerulus is excreted in urine following a massive and active tubular reabsorption [12]. Hence, aminoaciduria in diabetic individuals might be conditioned by the degree of structural and/or functional integrity of the

The aim of this study is to analyze amino acid plasma profile in a group of young diabetic individuals and to evaluate its potential application as markers of metabolic control of the disease, as well as to analyze the urinary excretion of amino acids in the absence of

A clinical assessment and metabolic study was accomplished in 49 children diagnosed with insulin-dependent diabetes mellitus, aged 8.6–14.3 years, following conventional insulin ther-

Information recorded from every patient/participant included age, weight and height, BMI,

Weight and height measurements were made in underwear while being barefoot. Weight was measured using the Año-Sayol scale (reading interval 0–120 kg and a precision of 100 g), and height was measured using the Holtain wall stadiometer (reading interval 60–210 cm, precision 0.1 cm). The Z-score values for the BMI were calculated using the epidemiologic data

apy, and a group of 48 healthy children (control group) aged 7.4–14.8 years.

time and progress of the disease, and dosage of subcutaneous insulin.

normal levels after intensive insulin therapy [3–6].

important due to its prognostic significance [7, 8].

renal tubule.

110 Diabetes and Its Complications

2.1. Participants

2.2. Clinical assessment

microalbuminuria in these patients.

2. Material and methods

All participants (diabetic and control group) underwent blood testing after a 12-hour fast, in order to determine plasma glucose levels, glycosylated hemoglobin (Hb1Ac), creatinine and amino acid concentrations. In addition, a 24-hour urine sample was collected to determine albumin and amino acid concentrations and glomerular filtration rate (GFR).

The analyzed amino acids (in blood and urine) were the following: alanine (ALA), arginine (ARG), aspartic acid (ASP), cysteine (CYS), glutamine (GLN), glutamic acid (GLU), glycine (GLY), histidine (HIS), isolecucine (ILE), leucine (LEU), lisine (LYS), methionine (MET), phenylalanine (PHE), serine (SER), threonine (THR), tyrosine (TYR), valine (VAL), and taurine (TAU).

Measurements in plasma (glucose and creatinine) and urine (creatinine) were made using a Synchron CX5 (Beckman) analyzer. HbA1c was determined using Boehringer-Mannheim reagents.

The quantification of urinary albumin excretion (UAE) was made by nephelometry (Away Protein System-Beckman), and microalbuminuria was considered when values exceed 12 ug/ min, being that a reason for exclusion. GFR was calculated using the endogenous creatinine clearance, and hyperfiltration was considered when values were over 145 ml/min/11.73 m2 .

The determination of urine and plasma amino acid concentrations was made by reversedphase high pressure liquid chromatography (HPLC) with o-phthaldialdehyde precolumn derivatization.

#### 2.4. Statistical analysis

Results are displayed as means (M) with corresponding standard deviations (SD). Statistical analysis (descriptive statistics, Student's T and Pearson's correlation) was done using the Statistical Packages for the Social Sciences version 20.0 (Chicago, IL, USA). Statistical significance was assumed when p value was lower than 0.05.

Parents and/or legal guardians were informed and provided verbal consent for the participation in this study in all cases. The study was approved by the Ethics Committee for Human Investigation at our institution (in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and later amendments).
