This observational study was conducted in a three month period (from July- September, 2015), after obtaining Ethical Clearances from the Institutes’ Ethical Committees. Total of 37 early neonates (0-3-day-old) (16 female) were selected for this study after obtaining signed informed consent from their parent or guardian.

Full term babies of 38-41 week gestational age (calculated from menstrual history and ultrasonography), birth weight 2500-4000 gram, and Apgar score ≥ 7 five minutes after birth were selected for this study.

Babies in the acute phase of physiologic jaundice and those currently taking any medications were excluded from the study.

Since only 37 babies were recruited in this study, they were not grouped on the basis of gender.

Spot urine samples were collected from the selected neonates by using paediatric urine collection bags. Urine collected in the bag was stored in non-sterile urine pot and taken to the laboratory for analysis. None of the samples contained blood (determined by using urine dipsticks) or faeces (observed visually).

Urinary microprotein was analysed using microprotein estimation kit (from Bio Lab Diagnostics) based on pyrogallol red dye binding method. Urinary creatinine was estimated using estimation kit (from Span Diagnostics) based on modified Jaffe’s method. The Protein Creatinine Ratio (PCR) of each sample was calculated from the equation:

The PCR values obtained from full term, normal early neonates were used to obtain the reference interval. Since the distribution was non-Gaussian, a non- parametric method was used. The 95% range (2.5^th–97.5^th percentile) was taken as the reference interval.

The protein and creatinine concentrations (mg/dL) in the random spot urine samples obtained from 0-1, 1-2, and 2-3-day-old infants are summarized in [Table/Fig-1]. The mean values of protein concentration do not show much variation in the three age groups, 13.67, 17.68, and 15.25 mg/dL, respectively. The average creatinine concentration was lower in 0-1-day-old infants (25.5 mg/dL) compared to creatinine excretion in infants older than one day.

Since there were only 6 infants aged 0-1 day, while there were nineteen 1-2-day-old babies and twelve 2-3-day-old babies, the results were not compared by ANOVA. Maximum variation in PCR was observed in 0-1 day old infants (0.11-0.82), compared to 1-2-day-old (0.29-0.78) and 2-3-day-old (0.17-0.33) infants.

The frequency curve [Table/Fig-2] for urinary protein creatinine ratio showed a median range of 0.4-0.6. The modal range (with maximum number of observations) was 0.3-0.4. Since the distribution of PCR values was non- Gaussian, the normal range of urinary PCR was determined by the non- parametric method (2.5^th - 97.5^th percentile) and was found to be 0.1-0.8.

[Table/Fig-2]:

Frequency distribution curve for urinary protein creatinine ratio in 0-3-day-old neonates

The protein concentration varied from 6-32 mg/dL in 0-3-day-old neonates. When the protein excretion was compared in infants of different ages (0-1 day, 1-2 day, and 2-3 day), the mean values did not show much variation: 13.667mg/dL (Day 0-1), 17.684 mg/dL (Day 1-2), 15.250 mg/dL (Day 2-3). Variation in protein excretion was less compared to that noted for creatinine [Table/Fig-1]. Creatinine is produced from creatine, which is almost exclusively located in the skeletal muscle. About 2% of the muscle creatine is converted into creatinine everyday, and is subsequently excreted in the urine [16]. Creatinine excretion varied from 14 to 65 mg/dL in 0-3-day-old neonates. Matos et al., have reported a high and variable creatinine excretion in the first few days of life in human neonates [17]. Although nephrogenesis begins at 5 weeks of gestation and is complete by 36 weeks of gestation, the GFR is very low as the nephrons are small and functionally immature [18]. The GFR increases after 36 weeks of gestation till birth, and shows a dramatic increase after birth due to increase in cardiac output and systemic blood pressure and decrease in renal vascular resistance leading to increased renal blood flow and effective glomerular filtration pressure. There is also an alteration in the pattern of renal blood flow distribution, an increase in the permeability of the glomerular membrane, and an increase in the surface area available for filtration [19]. Still the GFR in the full term neonate is very low (about 20 mL/min/1.73 m² increases to about 40 mL/min/1.73 m² at one month) [18], still less than the normal adult values (55-140 mL/min/1.73m2) [20]. The rapidly increasing values of GFR explain the high variation in creatinine excretion in early neonates.

Newborn babies have elevated plasma creatinine levels that are close to the maternal plasma creatinine values and do not reflect the size and muscle mass of the new born [21,22]. Since creatinine is a small molecule (molecular weight 113 dalton, radius 0.3 nm), it is able to pass through the placenta. Equilibration of maternal and fetal plasma creatinine levels occurs during the second half of gestation [23]. After birth, plasma levels of creatinine continue to remain high for about a week. This is due to the tubular reabsorption of creatinine, a result of back flow of creatinine across the leaky tubular and vascular structures. Once renal maturity is attained, creatinine excretion in urine increases and plasma creatinine levels fall to values normal for the age. Since the normal urine output in an early neonate is about ≥1mL/kg/h [3], creatinine excretion is slow and occurs over a period of about 7 days.

On comparing the creatinine excretion in infants of different age groups, the concentration of creatinine was less in 0-1-day-old infants (14-38 mg/dL, mean mg/dL). Concentration increased after the first day and was similar in 1-2 and 2-3-day-old infants [Table/Fig-1].

A comparison of the PCR values on days 0-1, 1-2, and 2-3 shows that PCR values showed a decreasing trend with the age of the infant [Table/Fig-1]. Also, maximum variation in the PCR values was observed on Day 0-1.

While the PCR values less than 0.2 are considered normal in children above 2 years of age and in adults, values up to 0.5 may be considered normal in 6-24 month old children [24]. This study suggests that random spot urinary PCR values of up to 0.8 may be considered normal in 0-3-day-old infants.

The size of study sample was small. Values of protein creatinine ratio obtained in one, two, and three-day-old infants could not be compared by ANOVA since there were only 6 infants in the 0-1-day-old category.

A larger study with more subjects needs to be conducted to study the variation in protein creatinine ratio in 1-7 day old full term and pre-term neonates. Reference intervals of PCR should be determined for narrow range of age groups in case of early neonates, e.g. 0-6h, 6-12h, 12-24h, 24-72h old babies.

Protein concentration in random spot urine sample from 0-3-day-old neonates varied from 3-32 mg/dL, while creatinine concentration varied from 14-65 mg/dL. Creatinine concentration was lower in 0-1-day-old infants. Protein creatinine ratio of the random spot urinary samples varied from 0.11 to 0.82. The ratio showed a greater variation in the 0-1-day-old infants. A PCR value of ≤ 0.8 may be considered normal for 0-3-day-old infants. Reference interval of PCR needs to be calculated for infants of different ages, e.g. 0-6 hour, 6-12 hour, 12-24 hour, 24-72 hour olds, since the values show a large variation due to rapidly changing glomerular filtration rate.