Urinary Lactate-To-Creatinine Ratio during the First Days of Life Correlates with the Degree of Brain Damage in Premature Infants

Currently, the most common neonatal brain injuries are those associated with preterm birth. In particular, infants born at <32 weeks of gestational age (very low gestational age [VLGA]) are at risk of adverse neurodevelopmental outcomes. This is mainly because such prematurity predisposes infants to structural brain injuries, such as perinatal white matter injury and intraventricular haemorrhage. The mechanisms associated with the development, course, and recovery after preterm brain injury and their impact on the disruption of further development of the immature brain are inadequately understood.

Brain hypoxia-ischaemia is considered one of the key players in the development of neonatal brain damage [1]. Lactate is the main end product of anaerobic glucose metabolism, and it has been established as a plasma marker of altered tissue perfusion and oxygen depletion in critically ill patients [2, 3].

Lactate, which is physiologically present in the plasma at low concentrations, is further metabolized by the liver, myocytes, and proximal tubule cells and, to a lesser extent, by renal excretion [4]. Insufficient tissue oxygen concentrations block the production of ATP during the citric acid cycle and result in increased ATP generation from significantly less energy-efficient anaerobic glycolysis, with concomitant lactate overproduction. In neonates, higher blood lactate levels are associated with a greater risk of mortality and morbidities, including neurological deficits [5]. Interestingly, lactate is a substrate for the preterm brain and may support brain energy metabolism [6].

Urine can serve as a valuable source of information for understanding metabolic alterations associated with severe perinatal outcomes. Urine sample collection is easy to perform, non-invasive, and free from clinical risk. The variation of urine constituents is key for homoeostatic control, and fluctuations in urine composition reflect systemic changes. Urinary lactate has been studied in hypoxic-ischaemic encephalopathy in term-born newborns [7, 8] and has proven informative. However, changes in the urinary lactate concentration in the context of preterm brain injury are insufficiently understood. One urinary metabolome study revealed elevated energy intermediates, including lactate, in preterm infants with IVH on the 1st day of life (DOL) [9]. However, this study included only 7 patients. Moreover, increased levels of lactate were found on the 2nd DOL in extremely preterm infants, and magnetic resonance imaging (MRI) at term-equivalent age (TEA) revealed moderate to severe white matter damage. However, the urinary lactate concentration was assessed only on the 2nd DOL and 10th DOL; therefore, the dynamics of urinary lactate concentrations are unknown [10]. Notably, data regarding the correlation between the lactate concentration in urine and the results of magnetic resonance spectroscopy (¹H-MRS), a valuable method to improve the evaluation of brain development after preterm birth [11], are lacking.

The aim of the present study was to investigate the evolution of urinary lactate concentrations in VLGA infants with and without preterm brain injury, assessed according to the Kidokoro score, during the first week of life, after 1 month of life, and at TEA. Moreover, we evaluated the associations between the urinary lactate-to-creatinine ratio (ULCR) and the concentrations of selected neurometabolites assessed via brain ¹H-MRS at TEA.

The enrolment criteria included consecutive infants born at <32 weeks of gestational age and admitted to the neonatal intensive care unit (NICU) within their first 24 h of life at Jagiellonian University Medical College (JUMC), Krakow, Poland, between April 2021 and July 2023. The exclusion criteria were as follows: (1) congenital anomalies of the heart/kidney or any structural abnormalities on cranial ultrasound on admission, (2) multiple pregnancies, and (3) a clinical suspicion of metabolic/genetic disorders. The Local Ethical Committee approved the present study, and written informed consent was obtained from the infants’ parents. Perinatal and neonatal demographic and clinical characteristics were prospectively recorded.

Urine samples for lactate and creatinine analysis were collected at eight time points: at the 1st, 2nd, 3rd, 4th, 6th, 8th, and 28th DOLs and at TEA (Fig. 1). Urine was collected non-invasively with a sterile cotton ball (Paul Hartmann, Pabianice, Poland) placed in a disposable diaper, from which the urine was aspirated with a sterile syringe (B. Braun Medical AG, Sempach, Switzerland) and transferred to a sterile polystyrene test tube (FL Medical, Torreglia, Italy). In the case of urine collection for medical reasons via a sterilized urine bag (Zarys, Zabrze, Poland), some of the urine obtained was aspirated from the bag into a sterile vial, using the same sterile syringe described above. Any urine sample contaminated with stool was discarded, and a new sample was collected. Upon collection, the urine was immediately transferred to the Department of Clinical Biochemistry, Institute of Paediatrics, JUMC.

The urine creatinine concentration was measured by a dry chemistry analyser (Vitros 4600, Ortho Clinical Diagnostics Inc., Rochester, NY, USA). Then, the urine was centrifuged for 10 min at 2,600 × g at 4°C and kept at −80°C until analysis. Urinary lactate was measured via highly selective liquid chromatography-tandem mass spectrometry in ESI-negative ionization mode (LC‒MS/MS, 1260 Infinity II, 6460 QTRAP; Agilent Technologies, Waldbronn, Germany) with a quantitative organic acid analysis kit (Zivak Technologies) and expressed as the ULCR. Each urine sample was pre-treated manually.

Fifty microlitres of reagent 1 was added to 300 μL of urine in a preparation tube, and the mixture was vortexed for 5 s. Then, 50 μL of the reagent 2–3 mixture was added, and the mixture was vortexed again for 5 s. The prepared sample volume injected into the LC‒MS/MS system was 20 μL. Specifically, the same pre-treatment procedure was used for the calibrators and control samples. The calibration curves were generated with 3 calibrators run in duplicate.

Sample separation was performed in reversed-phase mode on an Organic Acid Urine LC‒MS/MS Analytical Column (Zivak, Turkey). The gradient elution conditions for mobile phases A and B were as follows: 0 min at 100% A, 0.1–14 min at 100% B, 14.1–16 min at 100% B, 16.1–20 min at 100% A, and a flow rate of 0.25 mL/min. The column temperature was maintained at 35°C. Data processing was performed via Mass Hunter Workstation Software Quantitative Analysis v.B.09.00 (Agilent Technologies). To correct for differences in the absolute amount of water, lactate concentrations were related to urinary creatinine concentrations.

All study participants underwent conventional MRI and ¹H-MRS at TEA (37–42 weeks of post-menstrual age [PMA]). MRI and ¹H-MRS were performed using the 1.5T GE Optima 450w whole-body magnetic resonance scanner with a 20-channel head coil.

Details regarding the MRI examination are presented in the online supplementary materials (for all online suppl. material, see https://doi.org/10.1159/000542793).

¹H-MRS was performed using single-voxel spectroscopy and multivoxel techniques (2D chemical shift imaging). MRS examination was based on the PRESS technique (point-resolved spectroscopy sequence). The PRESS sequence utilizes a 90° and two 180° radiofrequency pulse. The Chemical Shift Selective Imaging Sequence (CHESS) was implemented for water suppression with a frequency-selective 90° pulse and dephasing gradient to destroy the water signal. The acquisition parameters of ¹H-MRS were TE 35 ms and 144 ms, TR 2,000 ms, 64 averages acquired. Two echo times (TE) were selected to record metabolites with both long and short relaxation times. The local magnetic field homogeneity was optimized by auto-shim procedure. The quality of the shimming obtained in the voxel was controlled by the spectral line width (full width of half maximum [FWHM] in Hz) of the unsuppressed water, obtained by the automated optimization sequence before scanning. The FWHM and signal to noise ratio values were used to exclude low-quality MR spectra. The spectra for FWHM <10 MHz and SNR above 90 were analysed.

Two-dimensional T2 imaging was performed to visualize anatomical brain structures and planning volume of interest position for spectroscopy. The volume of interest size was adjusted to the anatomical size of the thalamus and was approximately 1.69 cm³.

¹H-MRS data were analysed using SAGE software (Spectroscopy Analysis by GE) [7] and on AW (Advantage Workstation, GE) 4.5 workstation with Functool. The following metabolites were manually selected from the spectrum: lactates (Lac) (1.33 ppm), N-acetyl aspartate (NAA) (2.02 ppm), creatine (Cr) (3.02 ppm) (Fig. 2).

Once peaks were selected, SAGE software automatically calculated the peak area that correlates with metabolite concentration in the VOI. MRI of the brain, ¹H-MRS, and spectrum analysis were performed by a radiologist and medical physicist with over 10 years of experience. Metabolite concentrations were compared for the same test parameters, i.e., the same TE. A TE of 35 ms has been selected. The ratios of individual metabolite concentrations to each other were calculated.

All MR images were analysed twice according to the Kidokoro grading system [12]. Analyses were performed by four pediatricians and neonatologists (P.K., M.Z., A.K., and M.O.) who were previously trained by a neonatal neuroradiologist with >30 years of experience (I.H.S.), using the program RadiAnt DICOM Viewer. Readers were blinded to long-term outcomes and clinical data except for gestational age and PMA. Any disagreements in grading were resolved by consensus with neonatal neuroradiologist. Patients were divided into 3 subgroups (a global brain abnormality score (GBAS) indicating normality, a GBAS indicating mild brain damage and a GBAS indicating moderate + severe brain damage) according to the extent of premature brain injury.

Statistical analysis was performed with SPSS 29.0 software (IBM Corp.). Qualitative values were compared via Fisher’s exact test or the chi-square test, as appropriate. To assess the differences in continuous variables between the studied groups, the Mann-Whitney U test or Kruskal-Wallis test was used. Correlations were analysed by the Spearman test. Differences were found to be statistically significant if the probability of type I error alpha value was lower than 0.05.

A total of 54 patients were included in the study. The group of premature neonates was further divided into three subgroups according to the degree of brain damage determined via the Kidokoro grading system: no brain damage (GBAS indicating normality, 32 patients), mild brain damage (GBAS indicating mild brain damage, 11 patients), and moderate + severe brain damage (GBAS indicating moderate + severe brain damage, 11 patients). MRI + ¹H-MRS was performed at a median postnatal age of 66 days (Q1; Q3 54–75 days), and the median PMA was 38 weeks (Q1; Q3 37–40 weeks).

No differences in gestational age, birth weight, Apgar score, pH, or lactate level at 1 h of age were observed among the normal, mild, and moderate + severe subgroups. The median base deficit was significantly greater in the moderate + severe brain damage subgroup than in the normal subgroup (−10.7 mm vs. −4.1 mm; p = 0.001) and the mild brain damage subgroup (−10.7 mm vs. −4.4 mm; p = 0.02). The clinical characteristics of the patients are presented in Table 1.

Serial determination of the ULCR in the normal and mild brain damage subgroups revealed a decrease in the lactate concentration over time. However, in the moderate + severe brain damage subgroup, there was a marked increase in the ULCR on the 3rd DOL. In the following DOLs, in the moderate + severe brain damage subgroup, the lactate concentration gradually decreased (Fig. 3). Initial (1st DOL) lactic acid excretion did not differ significantly among the study subgroups. However, there was a trend towards a higher ULCR with increasing brain damage severity.

On the 2nd DOL, we observed a higher median ULCR in the moderate + severe brain damage subgroup than in the normal subgroup (39.85 vs. 16.88; p = 0.023). On the 3rd DOL, the median ULCR in the moderate + severe brain damage subgroup was greater than that in both the normal (122.13 vs. 12.07; p = 0.001) and mild (122.13 vs. 25.57; p < 0.001) brain damage subgroups. The levels of lactate in further collected urine samples (collected on the 4th DOL, 6th DOL, 8th DOL, and 28th DOL and at TEA) did not differ significantly among the subgroups (online suppl. Table 1).

Moreover, to assess the influence of gestational age on the ULCRs obtained, an additional analysis was performed considering the longitudinal nature of the measurement of the ULCR. Due to the nature of the distribution of variables, a generalized linear mixed model was used. Time, gestational age, serum lactate, and Kidokoro score were served as covariates. The results of the multivariate analysis confirmed that the ULCR differs significantly in the first four DOLs between groups of patients distinguished according to their future Kidokoro score, also after adjusting for the gestational age of the newborns.

An analysis of the ¹H-MRS results revealed that the GBAS moderate + severe brain damage subgroup had the lowest NAA/Cr ratio (statistically significant in the right thalamus) and the highest Lac/Cr and Lac/NAA ratios (statistically significant in the left thalamus) (Fig. 4).

We found a statistically significant correlation between the ULCR determined on the 3rd DOL and the Lac/Cr (r = 0.308; p = 0.022) and Lac/NAA (r = 0.334; p = 0.013) ratios in the right thalamus determined at TEA via ¹H-MRS. Considering the left thalamus, the correlation coefficients remained positive but were slightly smaller than those for the right thalamus, and these correlations were not statistically significant. On subsequent days of life, we did not observe correlations between the ULCR and the neurometabolite ratios (Table 2).

As a result of finding significant correlations between ULCR in the 3rd DOL and the ratios of selected neurometabolites (Lac/NAA and Lac/Cr) in the ¹H-MRS examination, we performed an additional multivariate analysis to correct the results in relation to the gestational age of the patients. The analysis showed that the measurement of ULCR in the 3rd DOL was an independent predictor of the Lac/NAA and Lac/Cr ratios measured in the right thalamus (corrected p values 0.038 and 0.009, respectively).

The results of this study demonstrated that VLGA infants with a GBAS indicating moderate + severe brain damage had higher ULCRs on the 2nd DOL and 3rd DOL than did those without brain damage and/or with mild preterm brain injury. Moreover, we showed that only the subgroup with moderate + severe brain abnormalities presented with a secondary increase in the urinary lactate concentration at the 3rd DOL, whereas in patients with less severe/no brain damage, the ULCR oscillated around similar values or gradually decreased. Additionally, we found a correlation between the ULCR collected on the 3rd DOL and the Lac/Cr and Lac/NAA ratios measured via ¹H-MRS at TEA.

Several exceptionally high ULCR values, present on the 1st DOL mainly in newborns from the moderate + severe brain damage subgroup, can be explained either by renal immaturity and increased tubular creatinine reabsorption [13] or by acute antepartum asphyxia [14], possibly indicated by higher initial base deficits.

Owing to the serial determination of lactic acid concentrations in urine within the first week of life, we determined that VLGA newborns with a GBAS indicating moderate + severe brain abnormalities presented with higher ULCRs on the 2nd and 3rd DOLs than those without and/or with mild brain injury. Additionally, a secondary increase in lactate excretion was detected on the 3rd DOL. This pattern of prolonged increased lactate excretion is alarming, as previous studies have shown associations between persistently increased or worsening lactate levels and adverse neonatal outcomes [15, 16]. Our results agree with those of Tataranno et al. [10], who reported that on the 2nd DOL, urinary lactate levels were significantly higher in infants born extremely preterm with moderate + severe white matter injury assessed according to Kidokoro than in those born preterm with normal or mildly abnormal white matter. Interestingly, in studies on full-term newborns with HIE, the concentration of lactic acid in urine was the highest on the 1st DOL and then gradually decreased [7]. As tissue hypoxia was the most frequent cause of increased lactate production experienced in the intensive care nursery [17], we can assume that in VLGA newborns with a GBAS indicating moderate + severe brain damage in the early postnatal period, there was a shift towards anaerobic metabolism and mitochondrial dysfunction, which was reflected in the ULCR results. Notably, the acceleration of anaerobic glycolysis may serve as a protective mechanism against hypoxia to guarantee the supply of oxygen and energy to the brain [18]. However, increased ULCRs in patients with moderate + severe brain damage may also be at least partially caused by altered liver and renal tubular metabolism [19] or action of catecholamines [4].

Analysis of the ¹H-MRS results revealed the accumulation of lactate in the thalami of infants with moderate + severe brain damage. This suggests ongoing inflammation or other subnormal developmental process. Notably, elevated Lac/Cr and Lac/NAA ratios in deep grey matter nuclei may be predictors of impaired neurodevelopmental outcomes [20]. Interestingly, according to our results, the urinary lactic acid concentration measured on the 3rd DOL was positively correlated with the brain Lac/Cr and Lac/NAA ratios assessed at TEA. To our knowledge, no study has yet demonstrated the existence of this correlation. The observed correlation underlines the importance of tissue hypoxia/ischaemia and alterations in energy metabolism occurring during the first DOL as possible causes of preterm brain injury.

This study has several strengths. The serial collection of urine samples from VLGA infants at multiple time points not only enabled us to trace the evolution of the ULCR up to TEA but also made it possible to precisely determine that the urinary ULCR is greater in patients with moderate + severe neonatal brain injury than in patients with no/mild brain injury and that it is the highest around the 3rd DOL. Moreover, we divided our patients into subgroups according to the degree of brain damage assessed according to the Kidokoro score, which was designed for premature infants, considers damage to the white matter, cerebral cortex, subcortical grey matter, and cerebellum, and has prognostic value [21]. Our study draws attention to ULCR as a potential non-invasive biomarker for detecting preterm brain injury. This approach could provide clinicians with a cost-effective, easily accessible method for assessing the risk/presence of brain damage in VLGA infants during critical early development stages. In our study, we used the LC-MS/MS method to determine the level of lactic acid in urine, which is not available in everyday diagnostics. Perhaps these samples could be evaluated under standard laboratory conditions, which would be clinically relevant, but for this purpose commercially available analysers that evaluate the level of lactic acid in other body fluids, such as blood or cerebrospinal fluid, would need to be validated. The major limitation of our study was the relatively small number of patients, especially in the subgroup with moderate + severe brain injury. The nature of the study was primarily explorative, and the statistical hypothesis was not initially defined.

Our results indicate that VLGA infants whose MRI at TEA reveals moderate + severe brain damage experience energy catastrophe early in life. Additionally, we also showed that this process is observed around the 3rd DOL in the form of the increase in the ULCR. The observed correlation underlines the importance of tissue hypoxia/ischaemia and alterations in energy metabolism during the first DOL as possible causes of preterm brain injury.

The author would like to thank the NICU staff and the anaesthesiologist Tomasz Langie at University Children’s Hospital in Krakow for their dedication to paediatric patients.

This study protocol was reviewed and approved by the Jagiellonian University Bioethical Committee, Krakow, Poland (Approval No. 1072.6120.336.2020). Written informed consent for participation in the study was obtained from the participants’ parents.

The authors have no conflicts of interest to declare.

The present study was supported by the National Science Centre, Poland (Grant No. 2020/37/B/NZ5/01099). The funder had no role in the design, data collection, data analysis, or reporting of this study.

Magdalena Zasada: conceptualization/design, methodology, investigation, data curation, analysis, and drafting of the initial manuscript. Marta Olszewska, and Aleksandra Kowalik: investigation, data curation, and review or editing of the manuscript. Joanna Berska, Joanna Bugajska, and Paulina Karcz: methodology, investigation, data curation, and review or editing of the manuscript. Izabela Herman-Sucharska: conceptualization/design, methodology, supervision/oversight, and review or editing of the manuscript. Przemko Kwinta: conceptualization/design, methodology, analysis, supervision/oversight, and review or editing of the manuscript.

All the data analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.