Establishment of reference intervals for urinary biochemical parameters: morning urine as a reliable alternative to 24-hour collection
Highlight box
Key findings
• Creatinine-normalized morning urine demonstrated strong correlation with 24-hour urinary excretion for multiple key biochemical analytes, including uric acid, urea, potassium, glucose, total protein, and α-amylase.
What is known and what is new?
• Twenty-four-hour urine collection is the gold standard for quantitative urinary biomarker assessment but is burdensome and prone to incomplete collection. Spot urine—particularly morning samples—is widely used in practice, yet comprehensive evidence on its agreement with 24-hour excretion across a broad panel of routine clinical analytes, with and without creatinine correction, is limited.
• This study established sex-specific reference intervals for 15 urinary analytes in three common specimen types and demonstrated that creatinine correction markedly enhances the concordance between morning spot urine and 24-hour urine.
What is the implication, and what should change now?
• Creatinine-adjusted morning urine can serve as a reliable, practical alternative to 24-hour collection for monitoring several clinically relevant urinary biomarkers. Laboratories and clinicians should consider adopting creatinine-normalized morning urine for routine assessment—especially when 24-hour collection is unfeasible—while using the provided reference intervals to guide interpretation. Local validation is recommended before implementation.
Introduction
Urine is a biological fluid widely applied in clinical examinations, playing a key role in disease detection, diagnosis, and health monitoring (1). Urine offers unique advantages compared to blood. First, urine tests are completely noninvasive, allowing for frequent and repeat sample collection (2). Second, urine proteomes are less complex than blood proteomes, making urine an optimal sample for proteomic analysis (3,4). Urinary biochemical parameters serve as key references for disease diagnoses across various clinical departments, including nephrology, urology, and hematology. For instance, through examining the concentration of certain chemistries in urine, including glucosuria, proteinuria, and inorganic salts such as urate, renal function can be monitored (5,6). Additionally, internal environment homeostasis can be evaluated through assessing the concentration of certain ions in urine, including Ca, Na, and K (7,8). As a part of routine medical examinations, urinary tests are low cost and suitable for large-scale population screening. They can also facilitate early disease detection, often before the appearance of distinct symptoms, thus improving prognosis.
Three types of urine specimens are commonly used in urinary biochemical tests: morning urine, random urine, and 24-hour urine. Among them, 24-hour urine is considered the gold standard (9) since it provides an overall excretion profile over the course of a day. However, collecting 24-hour urine samples can be challenging, especially for pediatric patients with low compliance or critically ill patients with dysuria (10). In contrast, morning urine and random urine are easier to collect and thus are ideal for rapid screening or routine medical examinations.
Reference intervals are fundamental standards for interpreting test results and making diagnoses. Accurate reference intervals provide a reliable laboratory basis for the clinical diagnosis and treatment of related diseases (11,12). However, no unified industry standard exists for the reference intervals of urinary biochemical parameters. The majority of laboratories rely on reference intervals provided by equipment or reagent manufacturers. Additionally, the influence of gender and age on the results is often overlooked. Therefore, we conducted a study with the aim of establishing reference intervals for urinary biochemical parameters across different types of urine based on healthy adults, providing a foundation for the diagnoses of relevant diseases. Furthermore, the correlation between various types of urine was analyzed to assess whether morning or random urine can serve as reliable alternatives to 24-hour urine for clinical evaluation. We present this article in accordance with the STROBE reporting checklist (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2026-1-0004/rc).
Methods
Study design and setting
A cross-sectional, observational study was conducted at The First Affiliated Hospital of Zhejiang Chinese Medical University. The reagents and instruments were acquired from Mindray Bio-Medical Electronics Co., Ltd. (Shenzhen, China).
Participants
A total of 240 healthy volunteers were recruited. After individuals with incomplete information and outlier age data were excluded, 230 adults (115 males and 115 females; age range, 20–50 years) were included in the final analysis. This study was approved by the Medical Ethics Committee of Zhejiang Provincial Hospital of Chinese Medicine (No. 2025-KLS-022-01). All participants provided written informed consent. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Volunteers aged 20 to 50 years with a perceived healthy body condition and no acute or chronic diseases were included. The exclusion criteria were as follows: (I) individuals who had undergone operations within the previous 6 months or who had experienced blood donation, blood transfusion or significant blood loss within the previous 4 months; (II) individuals with malnutrition, a long-term vegetarian lifestyle, alcoholism (defined as long-term or excessive alcohol consumption within 2 weeks), or smoking (defined as consuming more than 20 cigarettes per day); (III) individuals with cardiovascular diseases, metabolic diseases, nutritional diseases, repository diseases, urologic diseases, endocrine system diseases, aerodigestive diseases, rheumatic diseases, hematologic diseases, infectious diseases, tumors, or women who were pregnant or menstruating; and (IV) individuals who failed urine tests, were outside the specified age range, or provided incomplete information.
The sample size of this study (a total of 230 healthy adults, with 115 males and 115 females) was determined based on the feasibility of the prospective paired-design study and its primary research objective (to assess the correlation among different urine types). Although this was slightly below the ideal threshold of 120 individuals per subgroup recommended by the Clinical and Laboratory Standards Institute (CLSI) EP28-A3c guidelines for nonparametric methods, recent studies indicate that robust reference intervals can be established with smaller sample sizes through use of novel models based on biological variation data or the bootstrap method (13). Furthermore, our sample size exceeded the minimum thresholds (i.e., 60 individuals) demonstrated to be stable and reliable in multiple studies, providing an adequate statistical basis for the detection of key gender differences and calculation of precise confidence intervals.
Data measurement
We have substantially revised this paragraph: “A BS-2800M automatic biochemistry analyzer (Mindray) and its associated reagents were used. Creatinine (CREA) was measured using the compensated Jaffe method, traceable to an isotope dilution mass spectrometry (IDMS) reference method. Glucose (GLU) was measured by the hexokinase method, uric acid (UA) by the uricase-peroxidase method, and urea by the urease-glutamate dehydrogenase method. Electrolytes (Na+, K+, Cl-) were measured by indirect ion-selective electrode (ISE), and Ca2+, Mg2+, and P by photometric methods. Microalbumin (mALB), RBP, β2-MG, and total protein (TP) were measured by immunoturbidimetry. α-amylase (α-AMY) was measured by an enzymatic method. Internal quality control (IQC) was performed daily using two levels of commercial control materials (Mindray). The laboratory participates successfully in the external quality assessment (EQA) program provided by the National Center for Clinical Laboratories (NCCL) in China, which covers all urine analytes reported”.
Statistical analysis
The normality of the data distribution was tested via the Kolmogorov-Smirnov test, performed with MedCalc v. 15.2.2 (MedCalc Software, Ostend, Belgium) and SPSS v. R27.0.1.0 (The R Foundation for Statistical Computing, Vienna, Austria). Intergender differences were compared with the Mann-Whitney test. Reference intervals for biochemical parameters were established via nonparametric methods. The correlations between the three types of urine tests (24-hour, morning, and random) were evaluated via Spearman correction.
All biochemical parameter data in this study were derived from a prospectively collected standardized protocol, resulting in high data integrity. In the specific analyses, the overall missing rate for outcome data was less than 2%, primarily due to assay failure in individual samples. We assumed the missing data mechanism to be missing at random (MAR) and conducted all statistical analyses using complete-case analysis. Furthermore, to ensure the robustness of the main conclusions, we performed sensitivity analyses for key assessments using the multiple imputation method; the results were consistent with the primary analysis.
This study addressed the potential biases through (I) standardized sample collection and processing procedures; (II) correction for CREA; and (III) gender-stratified analysis. (I) For standardized sample collection and processing, the study provided standardized collection containers, written instructions, and one-on-one guidance to all participants to unify the collection methods for 24-hour urine, morning urine, and random urine. All samples were processed under identical centrifugation conditions (3,000 rpm for 5 minutes) to remove cells and larger particulates. This procedure aimed to minimize information bias caused by inconsistencies in collection and preprocessing operations. (II) For the use of CREA correction, the concentration of each biochemical parameter was divided by the urinary creatinine concentration from the same sample to calculate the CREA-corrected ratio. This method was intended to adjust for variability due to differences in urine concentration or dilution, thereby reducing the resulting measurement bias. (III) For gender-stratified analysis, the Mann-Whitney test was used to identify biochemical parameters that exhibited significant gender differences. Gender-specific reference intervals were then established separately for the identified parameters. The purpose of this approach was to proactively control for gender as a potential confounding factor, thereby avoiding misclassification or spurious associations that could arise from pooled analyses.
Results
Participant characteristics
This study aimed to establish reference intervals for urinary biochemical parameters. A total of 240 potential healthy volunteers were recruited. Based on predefined stringent inclusion and exclusion criteria, 10 volunteers were excluded due to incomplete information or outlier data. Consequently, 230 eligible participants (115 males and 115 females, with an age range of 20–50 years) were included in the study (Figure 1). The median age of the participants was 32 years. All included participants successfully completed the standardized collection procedure for all three types of urine samples: morning urine, random urine, and 24-hour urine. Ultimately, data from all 230 participants were used to establish reference intervals and for subsequent correlation analyses. The median urine volume was 1.5 mL, with a range of 0.43 to 2.66 mL (Figure 2).
Establishment of reference intervals for different types of urine tests
In this study, biochemical parameters from 24-hour urine, morning urine, and random urine samples from healthy individuals were analyzed. Reference intervals for these tests were established for the different urine sample types. Significant differences between genders were observed for CREA, Ca, and UA test results (P<0.05), suggesting the necessity of establishing gender-specific reference intervals for these parameters. According to clinical value evaluation and statistical analysis, no gender-specific reference intervals were required for the other 12 parameters.
To account for the potential influence of urine concentration or dilution on analyte concentrations, which is a significant source of biological variability in spot urine samples, we applied CREA correction. Given the impact of urine volume on certain biochemical items, CREA correction was applied to the previously mentioned parameters. The results of each biochemical parameter were divided by the urinary creatinine concentration. After correction, mALB exhibited a significant gender difference in the urine albumin: CREA ratio (UACR). No gender-specific reference intervals were necessary for any of the other parameters (P<0.05).
To accurately assess the concentration distribution of biochemical parameters (both before and after CREA correction) in morning urine, 24-hour urine, and random urine, normality analysis was performed, and corresponding box plots were generated (Figure 3). The results indicated that before CREA correction, significant differences were observed in most parameters between 24-hour urine and morning urine, as well as between 24-hour urine and random urine (P<0.05). Meanwhile, no significant difference was found between morning urine and random urine.
The correlation analysis between different types of urine tests
The correlation between morning urine and 24-hour urine before CREA correction is shown in Figure 4. It was found that seven parameters, including Ca, Cl, Na, Mg, α-AMY, RBP, and β2-MG, exhibited weak correlations between morning and 24-hour urine (0.3<r<0.5), while no correlation was identified for the remaining parameters. After CREA correction, the correlation between morning urine and 24-hour urine significantly increased. Specifically, five parameters, including GLU, mALB, P, TP, and UA, which showed no correlation previously, exhibited weak correlations postcorrection. Additionally, moderate correlations were observed for three other parameters—α-AMY, Mg, and β2-MG—which showed weak correlations before correction. Furthermore, a moderate correlation where no correlation was observed previously was identified for two parameters: K and urea. Notably, six parameters, GLU, K, TP, UA, urea, and α-AMY, had a substantial increase in correlation coefficients between morning and 24-hour urine, exceeding 50% after CREA correction.
Discussion
Urine analysis is an essential component of clinical routine examinations, with three types of urine specimens commonly used in biochemical tests: morning urine, random urine and 24-hour urine. Each type of specimen serves a specific purpose. Morning urine is a concentrated sample and is typically used for qualitative tests, including microscopic examination of cells, urinary casts, crystals, and bacteria. Random urine is commonly used for emergency examinations, such as urinary amylase testing. In contrast, 24-hour urine is primarily used for quantitative analyses, as it provides a comprehensive excretion profile over a 24-hour period (14). In this study, reference intervals for 15 routine laboratory biochemical parameters were established for 24-hour urine, morning urine, and random urine samples from 230 healthy adults. Additionally, urinary CREA was selected as a baseline to mitigate the influence of urine volume on the results. This allowed for the establishment of reference intervals for the ratio of urinary biochemical parameters to urinary CREA.
To assess the accuracy of the established reference intervals, comparisons were made between the results of this study and the reference intervals suggested by the Mayo Clinic Laboratories Interpretive Handbook (15) and other relevant literature (16-19).
The comparison was conducted for 12 parameters (Na, K, Cl, Ca, Mg, P, CREA, UA, urea, TP, mALB, and RBP) from 24-hour urine and for CREA and β2-MG from random urine, provided by the Mayo Clinic Laboratories, without CREA correction. The results revealed that eight parameters (Na, K, Cl, Mg, P, CREA, urea, and RBP) in 24-hour urine had intervals aligned with those suggested by the Mayo Clinic Laboratories. Differences were observed for other parameters in 24-hour urine—including Ca, UA, TP, and mALB—and for random urine—including CREA and β2-MG—which showed discrepancies when compared to the Mayo Clinic Laboratories reference intervals. However, this study’s reference intervals for K, Na, Glu, TP, and mALB were consistent with findings from previous studies (Table 1). After CREA correction, further comparisons were made between the results of this study and six reference intervals established by the Mayo Clinic Laboratories, including Ca, Mg, UA, TP, mALB, and RBP. Consequently, five parameters, including Ca, UA, TP, mALB, and RBP, had corresponding intervals with those of the Mayo Clinic Laboratories. In summary, the results of this study aligned closely with the reference intervals proposed by the Mayo Clinic Laboratories Interpretive Handbook or other previous studies, including for the parameters of Na, K, Cl, Ca, Mg, P, CREA, urea, TP, mALB, RBP, and β2-MG. However, certain parameters, particularly urate, exhibited significant differences (Table 2).
Table 1
| Parameter | Sample | Unit | n | LL | 90% CI for LL | UL | 90% CI for UL | RI for Mayo | RI for other studies |
|---|---|---|---|---|---|---|---|---|---|
| K | 24-hour | mmol/L | 230 | 9.7028 | 6.5650–11.4870 | 67.7091 | 55.3070 to 78.8840 | – | – |
| Morning | mmol/L | 230 | 7.6756 | 5.4050–9.9090 | 92.247 | 73.8370 to 110.1830 | – | – | |
| Random | mmol/L | 230 | 6.1938 | 4.5030–8.7050 | 109.9224 | 102.9140–129.5740 | – | – | |
| 24-hour | mmol/24 h | 230 | 13.5755 | 6.6871–17.0198 | 93.6836 | 78.6296–134.2749 | ≥18 years: 16–105 mmol/24 h | 32–121 mmol/24 h (19) | |
| GLU | 24-hour | mmol/L | 225 | – | – | 0.6781 | 0.5474–0.7992 | – | – |
| Morning | mmol/L | 229 | – | – | 0.7375 | 0.6322–0.8838 | – | – | |
| Random | mmol/L | 225 | – | – | 0.9224 | 0.7951–1.0764 | – | – | |
| 24-hour | mmol/24 h | 225 | – | – | 0.8904 | 0.7962–1.2482 | – | 0.1–0.6 mmol/24 h (19) | |
| Cl | 24-hour | mmol/L | 230 | 44.3087 | 31.5120–50.4200 | 213.5817 | 206.5520–262.4200 | – | |
| Morning | mmol/L | 230 | 25.5172 | 19.5100–40.8020 | 241.3336 | 221.8230–290.1150 | – | ||
| Random | mmol/L | 230 | 21.2037 | 9.7870–26.0790 | 259.1091 | 239.3110–289.0240 | – | ||
| 24-hour | mmol/24 h | 230 | 62.6909 | 43.7726–73.7235 | 345.1352 | 304.2645–404.4614 | ≥18 years: 34–286 mmol/24 h | 46–256 mmol/24 h (19) | |
| CREA | 24-hour | μmol/L | 230 | 3,063.543 | 2,639.2337–3,576.3664 | 19,079.7213 | 16,923.5570–22,791.3519 | – | – |
| Morning | μmol/L | 230 | 3,571.1664 | 1,650.4247–4,265.3450 | 33,128.7692 | 30,340.9455–37,267.9149 | – | – | |
| Random | μmol/L | 230 | 1,517.5509 | 1,036.1042–2,168.9644 | 36,545.7597 | 32,623.1995–45,322.5439 | ≥18 years old: 16–326 mg/dL (1,414.4–28,814.4 μmol/L) | – | |
| 24-hour | μmol/24 h | Male 115 | 6,956.3617 | 6,307.7881–7,744.8150 | 28,586.8652 | 25,241.9253–32,159.3232 | Male: ≥18 years: 930–2,955 mg/24 h (8,221.2–26,122.2 μmol/24 h) | Male: 7,000–21,000 μmol/24 h (19) | |
| Female 115 | 4,667.9371 | 4,289.0040–5,136.8627 | 22,906.5095 | 19,106.5138–27,650.8376 | Female: ≥18 years: 603–1,783 mg/24 h (5,330.52–15,761.72 μmol/24 h) | Female: 7,000–14,000 μmol/24 h (19) | |||
| Ca | 24-hour | mmol/L | 230 | – | – | 6.1577 | 5.5962–7.1853 | – | – |
| Morning | mmol/L | 230 | – | – | 8.7198 | 8.0164–9.8178 | – | – | |
| Random | mmol/L | 230 | – | – | 8.9731 | 7.0304–10.4026 | – | – | |
| 24-hour | mmol/24 h | Male 115 | – | – | 9.9162 | 8.5659–11.4259 | Male: <250 mg/24 hours (<6.25 mmol/24 h) | Male: 1.09–9.00 mmol/24 h (17) | |
| Female 115 |
– | – | 9.5924 | 8.2525–11.0996 | Female: <200 mg/24 hours (<5 mmol/24 h) | Female: 1.48–7.51 mmol/24 h (17) | |||
| UA | 24-hour | μmol/L | 230 | 931.1914 | 756.4654–1,112.6276 | 4,631.7875 | 4,302.5704–5,229.4748 | – | – |
| Morning | μmol/L | 230 | 972.3157 | 601.0826–1,161.7526 | 6,322.4976 | 5,883.4332–7,045.9003 | – | – | |
| Random | μmol/L | 230 | 464.2946 | 218.7729–599.9518 | 6,924.1882 | 6,353.6989–7,700.2600 | – | – | |
| 24-hour | μmol/24 h | Male 115 | 1,732.9844 | 1,562.5166–1,925.8424 | 7,307.8028 | 6,542.9532–8,071.0244 | Male: ≥18 years: 200–1,000 mg/24 h(1,190–5,950 μmol/24 h) | – | |
| Female 115 | 1,340.3182 | 1,181.1627–1,534.2855 | 7,333.2948 | 6,487.8466–8,264.2338 | Female: ≥18 years: 250–750 mg/24 h (1,487.5–4,462.5 μmol/24 h) | – | |||
| α-AMY | 24-hour | U/L | 230 | 66.3123 | 54.6922–79.7581 | 463.63 | 393.9071–603.1174 | – | – |
| Morning | U/L | 230 | 82.5438 | 55.3607–118.5976 | 832.1022 | 714.5959–1,107.0412 | – | – | |
| Random | U/L | 229 | 38.7981 | 13.1194–57.2054 | 903.4253 | 801.4894–1,012.6177 | – | – | |
| 24-hour | U/24 h | 230 | 102.7281 | 71.4802–126.8610 | 830.4148 | 517.5186–993.7503 | – | 52–274 U/24 h (19) | |
| Urea | 24-hour | mmol/L | 230 | 89.3734 | 78.4472–104.2840 | 478.6452 | 431.0387–610.3704 | – | – |
| Morning | mmol/L | 230 | 80.165 | 54.5330–105.7971 | 614.2452 | 588.6131–639.8773 | – | – | |
| Random | mmol/L | 230 | 51.5214 | 36.2979–64.0607 | 610.4555 | 547.8428–665.3153 | – | – | |
| 24-hour | mmol/24 h | 230 | 139.0066 | 109.0416–149.7627 | 757.7162 | 605.9836–1,174.5834 | ≥18 years: 7–42 g/24 hours (116.9–701.4 mmol/24 h) | – | |
| TP | 24-hour | mg/L | 229 | – | – | 88.1737 | 74.3234–114.0947 | – | – |
| Morning | mg/L | 228 | – | – | 113.1638 | 101.5201–131.5804 | – | – | |
| Random | mg/L | 228 | – | – | 145.8239 | 135.2847–167.8692 | – | – | |
| 24-hour | mg/24 h | 229 | – | – | 131.8492 | 110.1741–156.7440 | ≥18 years: <229 mg/24 h | <140 mg/24 h (20) | |
| P | 24-hour | mmol/L | 230 | 5.5622 | 4.7965–5.9957 | 41.1188 | 33.6124–50.1678 | – | – |
| Morning | mmol/L | 230 | 6.4112 | 4.4182–7.9412 | 63.391 | 53.2408–77.2210 | – | – | |
| Random | mmol/L | 230 | 1.7944 | 1.5565–2.6213 | 44.572 | 41.3154–51.6362 | – | – | |
| 24-hour | mmol/24 h | 230 | 7.6522 | 6.4753–8.8035 | 64.5351 | 40.6050–107.2840 | ≥18 years: 226–1797 mg/24 h (7.232–55.707 mmol/24 h) | Male: 5.86–31.60 mmol/24 h. Female: 6.05–31.24 mmol/24 h (17) | |
| Na | 24-hour | mmol/L | 230 | 44.3594 | 30.9980–49.8560 | 228.3681 | 209.1670–238.8420 | – | – |
| Morning | mmol/L | 230 | 21.9811 | 15.3680–36.3340 | 258.2253 | 237.8760–276.8990 | – | – | |
| Random | mmol/L | 230 | 14.0917 | 5.5110–21.1600 | 225.3332 | 214.8190–243.5180 | – | – | |
| 24-hour | mmol/24 h | 226 | 63.8178 | 53.5887–72.2631 | 335.8282 | 310.3600–415.5872 | ≥18 years: 22–328 mmol/24 h | 43–257 mmol/24 h (19) | |
| mALB | 24-hour | mg/L | 229 | – | – | 13.3636 | 10.3514–18.0066 | – | – |
| Morning | mg/L | 228 | – | – | 15.6216 | 13.1776–18.2256 | – | – | |
| Random | mg/L | 227 | – | – | 33.4944 | 27.3633–41.9704 | – | – | |
| 24-hour | mg/24 h | 229 | – | – | 19.5446 | 16.9479–27.0099 | <30 mg/24 h | 2–21 mg/24 h (19) | |
| RBP | 24-hour | mg/L | 94 | – | – | 0.1664 | 0.1408–0.2069 | – | – |
| Morning | mg/L | 94 | – | – | 0.2759 | 0.2228–0.3992 | – | – | |
| Random | mg/L | 94 | – | – | 0.386 | 0.2772–0.5610 | – | – | |
| 24-hour | mg/24 h | 94 | – | – | 0.2503 | 0.2174–0.2816 | ≥18 years of age: <0.273 mg/24 h | – | |
| Mg | 24-hour | mmol/L | 95 | 1.3494 | 1.1897–1.5431 | 7.4173 | 6.4350–8.4985 | – | – |
| Morning | mmol/L | 95 | 0.8153 | 0.6210–1.0674 | 9.5323 | 8.3037–10.8007 | – | – | |
| Random | mmol/L | 95 | 0.2291 | 0.08988–0.4517 | 7.8974 | 7.0402–8.7727 | – | – | |
| 24-hour | mmol/24 h | 95 | 1.9879 | 1.6869–2.3566 | 9.9574 | 8.3605–11.7596 | ≥18 years: 51–269 mg/24 h (2.0960–11.0559 mmol/24 h) | Male: 1.41–8.56 mmol/24 h. Female: 0.90–6.77 mmol/24 h (17) | |
| β2-MG | 24-hour | mg/L | 93 | – | – | 0.1356 | 0.1151–0.1537 | – | – |
| Morning | mg/L | 93 | – | – | 0.2819 | 0.1900–0.3559 | – | – | |
| Random | mg/L | 93 | – | – | 0.5082 | 0.3764–0.6379 | ≤0.3 mg/L | – | |
| 24-hour | mg/24 h | 93 | – | – | 0.227 | 0.1809–0.2773 | – | – |
α-AMY, α-amylase; β2-MG, β2-microglobulin; CI, confidence interval; CREA, creatinine; GLU, glucose; LL, lower limit; mALB, microalbumin; RBP, retinol-binding protein; RI, reference interval; TP, total protein; UA, uric acid; UL, upper limit.
Table 2
| Parameter | Sample | Unit | n | Mean | Median | LL | 90% CI for LL | UL | 90% CI for UL | RI for Mayo | RI for other studies |
|---|---|---|---|---|---|---|---|---|---|---|---|
| K | 24-hour | mmol/g | 230 | 30.2626 | 26.6464 | 10.7365 | 6.2798–12.0218 | 65.1058 | 58.4156–90.5264 | – | – |
| Morning | mmol/g | 228 | 21.022 | 19.0686 | 6.6322 | 6.3788–8.9115 | 46.9286 | 41.2580–53.1319 | – | 11.5–76.99 mmol/g CREA (21) | |
| Random | mmol/g | 229 | 32.5879 | 30.2484 | 8.8724 | 7.5350–11.6006 | 67.3382 | 62.9812–86.6475 | – | – | |
| GLU | 24-hour | mmol/g | 225 | 0.295 | 0.2491 | – | – | 0.6245 | 0.5131-0.8861 | – | – |
| Morning | mmol/g | 229 | 0.2331 | 0.2046 | – | – | 0.3956 | 0.3528-0.4450 | – | – | |
| Random | mmol/g | 225 | 0.2589 | 0.2218 | – | – | 0.4996 | 0.4155–0.7218 | – | – | |
| Cl | 24-hour | mmol/g | 230 | 136.261 | 125.1099 | 40.1794 | 26.4651–53.8018 | 305.3299 | 275.7339–340.6757 | – | – |
| Morning | mmol/g | 230 | 88.9093 | 75.2292 | 21.8476 | 18.7895–27.8923 | 254.603 | 212.1215–341.5091 | – | 20.35–257.25 mmol/g CREA (21) | |
| Random | mmol/g | 229 | 102.9135 | 93.7362 | 26.605 | 18.5413–34.2920 | 245.6848 | 216.8047–328.2246 | – | – | |
| Ca | 24-hour | mmol/g | 229 | 3.296 | 2.8773 | 0.9602 | 0.7255–1.1719 | 8.5233 | 7.0650–9.6835 | – | – |
| Morning | mmol/g | 229 | 2.09815 | 2.05236 | 0.5218 | 0.2064–0.7114 | 8.316 | 6.7258–11.8374 | – | 0.62–10.35 mmol/g CREA (21) | |
| Random | mmol/g | 230 | 2.745 | 2.2523 | 0.4779 | 0.3021–0.6507 | 7.2868 | 6.5385–14.1909 | 18–83 years: 0.05–0.27 mg/mg CREA (1.25–6.75 mmol/g CREA) | – | |
| UA | 24-hour | μmol/g | 229 | 2,655.8767 | 2,591.8548 | – | – | 3,772.3994 | 3,644.4187–3,900.3801 | – | – |
| Morning | μmol/g | 229 | 2,125.3767 | 2,049.4857 | – | – | 3,303.5024 | 3,168.4605–3,438.5443 | – | – | |
| Random | μmol/g | 229 | 2,360.4154 | 2,326.7701 | – | – | 3,645.876 | 3,549.9856–4,104.6171 | ≥18 years: <0.60 mg/mg CREA (<3,570 μmol/g CREA) | – | |
| α-AMY | 24-hour | U/g | 230 | 214.5139 | 206.0787 | 109.566 | 76.3534–114.3661 | 381.4113 | 343.9453–462.9706 | – | – |
| Morning | U/g | 230 | 212.8939 | 205.2557 | 106.0755 | 96.9474–116.0251 | 379.9162 | 341.3251–482.2100 | – | – | |
| Random | U/g | 230 | 239.1292 | 227.4868 | 114.5257 | 81.5202–124.0858 | 449.389 | 415.8742–535.9557 | – | – | |
| Urea | 24-hour | mmol/g | 230 | 243.3701 | 235.7084 | 134.4087 | 127.0634–149.4668 | 415.9076 | 387.9902–453.0188 | – | – |
| Morning | mmol/g | 230 | 236.9029 | 233.1368 | 109.1843 | 93.9055–119.8939 | 412.4789 | 383.5203–492.8053 | – | 141.59–509.73 mmol/g CREA (21) | |
| Random | mmol/g | 230 | 229.8235 | 224.7436 | 101.6446 | 80.6561–108.9561 | 390.8122 | 366.9152–446.3413 | – | – | |
| TP | 24-hour | mg/g | 227 | 38.8009 | 34.7904 | – | – | 68.2118 | 60.9490–91.8988 | – | – |
| Morning | mg/g | 227 | 35.2966 | 32.0918 | – | – | 54.5122 | 50.0000–58.7191 | – | 32.74–116.8 mg/g CREA (21) | |
| Random | mg/g | 225 | 42.5232 | 39.1213 | – | – | 67.0853 | 62.3329–71.9889 | ≥18 years: <0.18 mg/mg CREA (180 mg/g CREA) | 30–135 mg/g CREA (19) | |
| P | 24-hour | mmol/g | 230 | 15.5184 | 15.3209 | 7.5802 | 5.0562–8.6540 | 26.1117 | 23.8355–29.4636 | – | – |
| Morning | mmol/g | 230 | 17.2244 | 16.5939 | 6.764 | 5.7600–7.7681 | 27.6847 | 26.6807–28.6888 | – | – | |
| Random | mmol/g | 230 | 13.169 | 12.3754 | 3.4353 | 2.3053–5.1480 | 27.2949 | 25.2344–37.9974 | – | – | |
| Na | 24-hour | mmol/g | 230 | 138.839 | 129.7214 | 41.6081 | 30.6486–58.2021 | 274.1739 | 245.6886–306.0119 | – | – |
| Morning | mmol/g | 230 | 94.3494 | 81.92 | 20.7735 | 12.5487–27.1502 | 280.5939 | 219.2170–372.8924 | – | 28.31–252.21 mmol/g CREA (21) | |
| Random | mmol/g | 230 | 92.3718 | 81.5879 | 17.3406 | 12.7868–22.8551 | 226.1098 | 203.2525–356.9768 | – | – | |
| mALB | 24-hour | mg/g | 229 | 5.4947 | 3.7674 | – | – | 15.9522 | 11.3985–22.4449 | – | – |
| Morning | mg/g | 229 | 4.2005 | 3.6007 | – | – | 9.0394 | 7.5940–11.1242 | – | 0.88–11.5 mg/g CREA (21) | |
| Random | mg/g | Male 114 | 7.1005 | 3.9743 | – | – | 18.8134 | 14.2298–23.0110 | Males: <17 mg/g CREA | 1.2–14.8 mg/g CREA (19) | |
| mg/g | Female 115 | 8.1325 | 5.0379 | – | – | 24.3214 | 15.0414–31.9837 | Females: <25 mg/g CREA | 1.9–19 mg/g CREA (19) | ||
| RBP | 24-hour | mg/g | 95 | 0.08717 | 0.09862 | – | – | 0.5247 | 0.1778–1.4835 | – | – |
| Morning | mg/g | 95 | 0.9746 | 0.07427 | – | – | 0.2702 | 0.1622–0.4404 | – | – | |
| Random | mg/g | 95 | 0.09599 | 0.08727 | – | – | 0.241 | 0.1914–0.2961 | ≥18 years: <0.190 mg/g CREA | – | |
| Mg | 24-hour | mmol/g | 94 | 3.8443 | 3.7676 | 1.4532 | 1.0934–1.8130 | 6.2354 | 5.8756–6.5952 | – | 1.06–7.168 mmol/g CREA (21) |
| Morning | mmol/g | 94 | 2.2128 | 2.3158 | 0.8095 | 0.6920–0.9563 | 6.3491 | 5.5240–7.3660 | – | – | |
| Random | mmol/g | 95 | 1.851 | 1.838 | 0.6942 | 0.6042–0.8027 | 4.9722 | 4.2760–5.6943 | 18–83 years: 0.04–0.12 mg/mg CREA (1.64–4.92 mmol/g CREA) | – | |
| β2-MG | 24-hour | mg/g | 95 | 0.05499 | 0.05592 | 0.005621 | 0.001793–0.01776 | 0.5564 | 0.1743–1.6904 | – | – |
| Morning | mg/g | 95 | 0.05711 | 0.05497 | 0.0125 | 0.008660–0.01812 | 0.2416 | 0.1604–0.3450 | – | – | |
| Random | mg/g | 95 | 0.06206 | 0.06111 | 0.01255 | 0.008587–0.01906 | 0.2976 | 0.1949–0.4370 | – | – |
α-AMY, α-amylase; β2-MG, β2-microglobulin; CI, confidence interval; CREA, creatinine; GLU, glucose; LL, lower limit; mALB, microalbumin; RBP, retinol-binding protein; RI, reference interval; TP, total protein; UA, uric acid; UL, upper limit.
The potential causes of these differences may include the following: (I) parameters can be influenced by various factors, including population characteristics, race, genetics, geographic location, lifestyle, and diet. Urinary biochemical parameters may vary due to differences in dietary habits and lifestyles across regions. For instance, diets with high levels of UA are closely related to elevated urate levels (17), which could explain the higher reference interval for urate observed in 24-hour urine. The participants in this study, consisting of healthy young and middle-aged adults (aged 20–50 years), were likely to have a relatively higher intake of UA in their diets. This might have contributed to the observed higher reference intervals for UA in 24-hour urine in both males and females compared to those in the Mayo Clinic Laboratories Interpretive Handbook and other studies. Regarding CA, the reference interval established in this was is higher than that established in the Mayo Clinic Laboratories Interpretive Handbook. However, it is relatively close to the reference intervals of CA in 24-hour urine established by Mai et al. (16), which are 1.09–9.00 mmol/24 h for males and 1.48–7.51 mmol/24 h for females. (II) The sample size of healthy adults in this study was relatively limited, which could have introduced sampling errors when compared to the general population. (III) Differences in laboratory procedures, including the methods used for assessment, reagents, equipment, and testing platforms, can contribute to variations in reference intervals. (IV) Despite rigorous inclusion and exclusion criteria, some volunteers might have had underlying health conditions that were not detected during the health screens. (V) Although all volunteers received one-on-one guidance on the proper sample collection, it was difficult to verify with certainty that all samples were collected according to the prescribed protocols. Thus, we recommend that laboratories verify the results prior to establishing reference intervals based on the results of this study.
Additionally, the ratio of urinary biochemical parameters to urinary CREA was calculated, and corresponding reference intervals were established. After CREA correction, the reference intervals of routine biochemical parameters in this study exhibited a higher degree of consistency compared with those provided by the Mayo Clinic Laboratories. The improvement can likely be attributed to the fact that CREA correction mitigates the influence of urine concentration and dilution on the biochemical parameters (21). Therefore, urine samples collected at different times such as random urine and morning urine are more comparable after correction, resulting in reference intervals that are more aligned with those of the Mayo Clinic Laboratories.
One significant finding in this study is that random urine had a lower upper reference limit and a wider reference interval compared to morning urine and 24-hour urine. This observation may be explained by the nature of random urine samples, which can be conducted at any point during the day, making them less controlled and more prone to natural fluctuations in urine composition.
In this study, the values measured in 24-hour urine were considered to be the gold standard. The correlations between 24-hour urine and morning urine, both before and after CREA correction, were assessed. Before CREA correction, the correlation between morning urine and 24-hour urine was relatively low. However, after CREA correction, the correlation significantly improved, particularly for the ratios of urine electrolytes and urine amylase to CREA. This increase in correlation could be due to the fact that morning urine, being more concentrated, reflects renal clearance and excretion functions during the night. These functions are more comprehensively captured in 24-hour urine samples. By correcting for the effects of urine concentration and dilution, we can enhance the reliability of morning urine as a surrogate for 24-hour urine samples, rendering it more suitable for quantitative examinations. Since morning urine is typically more concentrated due to longer bladder retention, applying CREA correction adjusts for this natural concentration, providing results that align more closely with those from 24-hour urine for quantitative examinations.
Conclusions
This study established reference intervals for urinary biochemical parameters in 24-hour urine, morning urine, and random urine, with gender disparities being taken into account. Data before and after CREA correction were both considered. By comparing the biochemical parameters of morning urine and 24-hour urine, especially after creatinine correction, we demonstrated that morning urine has considerable potential as an alternative to 24-hour urine in assessing urinary biochemical parameters. Thus, our findings hold significant implications for clinical diagnosis, treatment, and monitoring. In clinical settings where time or resources are limited, morning urine samples can serve as a reliable, convenient substitute for 24-hour urine samples for the completion of quantitative examinations. However, caution is warranted when extrapolating these results to other age groups, ethnic populations, individuals with specific metabolic conditions, or different analytical platforms, as factors such as diet, genetics, and assay methods may influence urinary analyte levels. Therefore, we recommend that clinical laboratories validate or adjust these reference intervals using local samples before adoption in accordance with guidelines such as those of CLSI EP28-A3c. This step is crucial for ensuring accurate interpretation of test results in a diversity of clinical contexts.
Acknowledgments
We would like to express our sincere gratitude to the study team members. Our heartfelt thanks also go to the workers who selected the participants based on the selection criteria, as well as to all the study participants who kindly agreed to take part in the study. We would also like to thank Mindray for providing the instrument and reagents necessary for this study.
Footnote
Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2026-1-0004/rc
Data Sharing Statement: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2026-1-0004/dss
Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2026-1-0004/prf
Funding: This work was supported by
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2026-1-0004/coif). Y.L., Y.G. and J.Y. are from Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China. The other authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Medical Ethics Committee of Zhejiang Provincial Hospital of Chinese Medicine (No. 2025-KLS-022-01) and informed consent was taken from all participants.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
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(English Language Editor: J. Gray)
Cite this article as: Wang Z, Shen Y, Wang X, Liu Y, Guo Y, Ye J, Zuo F, Hu Z. Establishment of reference intervals for urinary biochemical parameters: morning urine as a reliable alternative to 24-hour collection. J Lab Precis Med 2026;11:13.
