Challenges in Urinalysis

#### **Chapter 4**

## Role of Urine Examination in Renal Transplant Recipients

*Lovelesh K. Nigam*

#### **Abstract**

Kidney transplantation has emerged as a major advance of modern medicine, providing high-quality life years to patients with end-stage renal disease (ESRD). Post-transplant monitoring of the transplanted kidney is based on physical examination, urine volume, the assessment of albuminuria or proteinuria, serum creatinine, and glomerular filtration rate (GFR) estimation based on serum creatinine. Of these multiple investigations, serum creatinine and urine analysis is one of the most widely used and accepted tool to assess graft dysfunction as well as plan management. Various immunological (rejections-antibody, cellular) and nonimmunological (polyoma virus nephropathy, mycosis, recurrent/de novo diseases) may affect the graft function. Changes in various parameters like urine osmolality, proteinuria, hematuria and presence of casts, crystals and other cellular constituents aids in diagnosis diseases of the allograft. This chapter thus highlights the importance of most frequent parameters that help in assessing the graft function. In addition to these parameters, a brief introduction of biomarkers is also included. Many studies have shown that these biomarkers have a promising role in diagnosis of allograft disease and thus avoiding interventional procedures like renal biopsy. Easy availability as well as low-cost of the urine examination makes it a promising tool for overall assessment of the graft dysfunction.

**Keywords:** renal transplant, proteinuria, hematura, rejection, tubular injury, biomarkers

#### **1. Introduction**

Kidney transplantation has emerged as a major advance of modern medicine, providing high-quality life years to patients with end-stage renal disease (ESRD) [1, 2]. The prevalence of end-stage renal disease requiring transplantation in India is estimated to be between 151 and 232 per million population [3]. Post-transplant monitoring of the transplanted kidney is based on physical examination, urine volume, the assessment of albuminuria or proteinuria, serum creatinine, and glomerular filtration rate (GFR) estimation based on serum creatinine [4]. Of these multiple investigations, serum creatinine and urine analysis is one of the most widely used and accepted tool to asses graft dysfunction [3]. Urine examination, known as "Uroscopy" in ancient time was considered as the mirror of medicine for several thousands of years. The physicians felt they could view the body's inner workings and get the insight of

the disease process by urine examination [5]. Urine examination aids diagnosis as well as management of both native as well as allograft kidney diseases [6].

Specific patterns in urinalysis provide information about graft function as well as renal diseases that can influence graft function [7]. It is a readily accessible, noninvasive tool, can be repeated anytime, cost effective as well as better tolerated than an invasive renal allograft biopsy. There are various causes for graft dysfunction, and these could be either acute or late. Urine analysis can help in diagnosis, follow-up and as well help in determining the graft outcome. Urinary abnormalities, such as hematuria or casts, are also useful in detecting and diagnosing allograft dysfunction [8, 9].

#### **1.1 Causes of graft dysfunction**

Before discussing about the role of urinalysis, it is important to determine the reasons for renal allograft dysfunction [9, 10]. Renal allograft dysfunction may be acute or late, the causes can broadly be classified as immunological or non-immunological. The immunological causes are usually acute and chronic rejections. The non-immunological causes include recurrence of a native disease, infections (bacterial, viral or fungal), acute tubular injury, drug toxicity, vascular complications, etc. [10]

Various parameters have been analyzed in urine of renal transplant recipients. These include determination of urine volume, urine osmolality, protein, glucose, blood and leucocytes. We conducted a pilot study in 310 renal transplant recipients who underwent renal allograft biopsy over a period of one year, where we analyzed the corresponding urinary findings which were compared with the morphological findings on renal allograft biopsy.

#### **2. Urine osmolality**

Osmolality marks the renal concentrating power, which depends on tubular function of the nephrons. Mazloum et al. in their study observed that altered osmoregulation performance, three months after transplantation is independently associated with allograft loss as well as reduced mGFR at 12 months [11]. When the graft suffers an ischemic lesion, the osmolality is lower as compared to that of a healthy kidney [12]. When we analyzed our set of patients, we found that the mean osmolality for patients with morphological evidence of rejection on RAB was 322.7 ± 141.3 mOsmol/l. This value was high as compared to patients having biopsy that were unremarkable for any immune or non-immune injury (mean urine osmolality: 116.2 ± 75.2 mOsmol/l). The osmolality of patients with biopsy features of acute tubular injury was 210 ± 82.2 mOsmol/l. Overall we recorded a higher value for urine osmolality in patients having acute rejection as compared to acute tubular injury or an unremarkable graft morphology.

Similar findings were also reported by Jenni et al. The receiver operator curve for osmolaluria to predict a rejection in the first 14 postoperative days showed an AUC (area under the curve) of 0.816 on day 2. The same study observed that if osmolaluria falls below 600 mOsmol/l, sensitivity and specificity for prediction of rejection is 66.7% and 89.5%, respectively [7]. Otto Schuck et al. examined early-morning urine osmolality in 104 transplant recipients (aged 21–76 years) and compared with findings of chronic renal allograft nephropathy by studying changes of interstitial fibrosis and tubular atrophy on biopsy. They postulated that the concentrating capacity of the graft kidney is decreased, however they did not report a significant correlation between concentrating function and tubulointerstitial histology findings with a mean urine

osmolality of 384 ± 120 mOsmol/l [13]. In our patients with chronic renal allograft nephropathy the mean osmolality was found to be 282.4 ± 137.1 mOsmol/L. In biopsies with morphological features of interstitial fibrosis and tubular atrophy the mean urine osmolality was 242.2 ± 114.4 mOsmol/l. We conclude that alone urine osmolality might not be a good variable for diagnosis. The values need to be interpreted with respect to clinical features as well as taking other findings in considerations.

#### **3. Proteinuria**

Proteinuria (including albuminuria) is an independent factor implicated in kidney damage in native as well as kidney allografts [14]. Recommendations are to perform urinalysis and urinary protein excretion to be assessed regularly in the posttransplant period. Most of the studies recommend that these investigations need to be performed at least every 2 to 3 months during the first post-transplant year and annually henceforth [8]. Many unique proteins, peptides, and other substances are excreted in urine in the patients who undergo renal transplant which could be useful to predict the outcome of the renal allograft [15, 16]. It is estimated that proteinuria is a common finding in post-transplant patients, the incidence being more than 40% kidney transplant per year. Various studies have found that even if proteinuria is low (<500 mg/day), there is still significant reduction in the graft function and reduced patient survival [17]. Even late onset proteinuria in post-transplant patients has been found to be associated with reduced graft and patient survival [18]. Proteinuria in the first year of transplant appears to be multi-factorial. Common causes of proteinuria implicated are residual proteinuria, glomerular diseases, effects of anti-HLA class II antibodies and drugs like mTOR inhibitors, tubulointerstitial disease of the graft, nephrosclerosis, renal vein thrombosis and reflux nephropathy [7, 17]. Causes for late onset proteinuria in renal transplant patients include: relapse or de novo glomerulonephritis, transplant glomerulopathy and chronic rejections. A proteinuria of >0.5 g/l and > 0.8 g/l have found to have a specificity of 80% and 90%, respectively, regarding prediction of rejection [7]. Studies have shown pre-transplant proteinuria (even of nephrotic range) considerable reduces in the first weeks, once a normal functioning kidney is transplanted [7, 17]. This happens due to reduction in the blood flow which occurs in native kidneys after transplant, if the graft is functioning normally. In a patient with poor graft function, the blood flow of native kidneys is maintained, which is the cause for persistent proteinuria in such patients. For patients with a normal functioning graft, the presence of proteinuria above 3000 mg/day, three weeks after the transplant should raise a suspicion for presence of a glomerular disease. This could either be a de novo or a recurrence of a primary glomerulonephritis in the graft.

Many studies have studied the causes for post-transplant proteinuria. Among the commonly used immunosuppressive agents, only Sirolimus has been implicated in development of post- transplantation proteinuria [19]. One of the studies documented that 58% of transplant patients with proteinuria (150 mg/day) did demonstrate transplant-specific lesions (allograft nephropathy, transplant glomerulopathy, or acute rejection) on biopsy as compared to 11% of patients that showed morphological evidence of glomerulonephritis on biopsy [20]. Shamseddin et al. in his meta-analysis stated that allograft nephropathy was documented in 8–54% of patients (average: 32%). Transplant glomerulopathy ranged from 0 to 39% (average: 17%) with an average prevalence of 37% as compared to glomerular disease [21].

Various methods have been implicated in estimation of urinary protein. Of all the methods available, urine dipstick testing is highly specific and most commonly used method used in most of the laboratories. Despite of false-positive or false-negative results that can be obtained in some situations, it is still the most preferred screening method for proteinuria. As urine dip-stick is not as sensitive as quantitative methods, a twenty-four-hour urine protein excretion stands as the gold standard for quantitative protein assessment. In cases where a twenty-four hour urine collection is problematic, urinary protein/creatinine (mg/mg) ratio can be assessed in a 'spot' urine. A UPCR acts as an excellent surrogate and is shown to have an excellent correlation with the protein content of a twenty-four-hour urine collection [22].

In our study the mean 24-hour urinary proteinuria was highest in cases which presented with recurrence of the native disease (4.9 ± 2.31 g) followed by patients with biopsies showing chronic allograft nephropathy (2.69 ± 1.96 g). Proteinuria was insignificant in biopsies with acute rejection (0.5 ± 1.46 g). Of the recurrent diseases maximum proteinuria was observed in biopsies showing focal and segmental glomerulosclerosis (5.7 ± 3.8 g), followed by those with IgA nephropathy (4.4 ± 3.6 g) and membranoproliferative glomerulonephritis (4.5 ± 1.7 g). Thus evaluation by 24-hour urine protein does help in diagnosis of recurrent diseases as well as chronic allograft nephropathy.

#### **4. Glucosuria**

Recurrence of diabetic nephropathy in renal allograft and post-transplant diabetes mellitus are the main reasons for glycosuria. Multiple factors come into play for the above stated diseases. These include transplant done in an old aged patient, high body mass index, presence of a family history of diabetes, use of immunosuppressive reagents (Prednisone, Tacrolimus) and concomitant history of hypertension. Other risk factors include polycystic kidney disease, episode of immune injury (acute rejection), hepatitis B virus infection and hepatitis C virus infection. However the KDIGO guidelines recommend determination of blood glucose levels and glycosylated hemoglobin for diagnosis of diabetes, the role for the measurement of glucosuria after renal transplantation is limited [7, 23]. In our study of one year, we did not come across any case of glycosuria or post-transplant new onset diabetic nephropathy.

#### **5. Hematuria**

Presence of at least five red blood cells/high power field (hpf) in three of three consecutive centrifuged specimens obtained at least seven days apart is defined as hematuria [24]. Haematuria may be present in 0.7–3% of the general population, and has a much higher prevalence in patients undergoing renal transplant. Hematuria, like proteinuria has been implicated as one of the factors for graft loss [25]. Increased bleeding tendency in renal allograft recipients could be possibly due to preexisting states of postrenal transplant patients, the use of antiplatelet agents for cardiovascular disease and platelet dysfunction. Additionally, post-transplant patients are susceptible to anemia which accentuates bleeding diathesis. This usually occurs as the circulating red blood cells displace platelets towards the vessel wall thus leading to contact with the subendothelial tissue at the site of injury. Also red blood cells release

adenosine diphosphate which inactivates prostacyclin and enhances platelet function [25, 26]. Although mechanisms of hematuria are many, following causes are main causes for hematuria in renal transplant patients:

#### **5.1 Infections**

Immunosuppressants are mainstay for graft stability, however use of these agents predisposes patients to urinary tract infections, which can be heralded by the sign of haematuria. Rivera-Sanchez conducted a prospective study on post-renal transplant patients with hematuria. They reported nearly 37% of the renal transplant recipients with hematuria have urinary tract infection, of which 13.4% had history of recurrent infections [27]. Certain predisposing factors have been implicated in causing recurrent acute graft pyelonephritis. These include presence of anatomical abnormalities like strictures at the ureterovesical junction or neurogenic bladder. Vesicoureteral reflux in these patients also contribute to recurrent infections [24]. As these patients are immunosuppressed, a higher index of suspicion for mycobacterial, fungal, and viral infection has to be kept in mind. Hematuria can occur secondary to cystitis, sparing the kidneys and can be associated with bacteria, fungus or viruses. Fungal organisms associated with hemorrhagic cystitis include *Candida albicans*, Cryptococcus, Aspergillus fumigates and mucormycosis whereas viruses implicated include BK virus, adenovirus, Cytomegalovirus, and herpes virus [28, 29].

#### **5.2 Malignancy**

Patients undergoing renal transplant are at risk of developing certain malignancies, in particular those cancers that are associated with viral infections. Common viruses include human papillomavirus (HPV) for cutaneous malignancies and Epstein–Barr virus (EBV) which are associated with post-transplant lymphoproliferative diseases.

Incidence of urological malignancies in these patients is less common. However some malignancies that can occur in these patients and present as hematuria include, renal cell carcinoma and cancers of the urinary bladder. Risk factors implicated in development of renal cell carcinoma are: prior history of renal cell carcinoma, polycystic kidney disease (PKD), duration of dialysis pre-transplant and tuberous sclerosis [30]. Larcom et al. showed that there is an estimated twofold increase for development of prostate carcinoma in the first 3 years after transplantation [31].

#### **5.3 Rejections**

Chronic rejection of the transplanted kidney typically presents with microscopic haematuria. Isolated case reports of patients with rejection presenting with gross haematuria have been documented [32].

#### **5.4 Disease recurrences**

Haematuria is a common manifestation of recurrence of glomerulonephritis. Those glomerulonephritis which present with a primarily nephritic picture present with hematuria predominantly. These commonly include Goodpasture's syndrome, systemic lupus erythematosus, and Ig A nephropathy. Acute syndromes that present with hematuria and lead to acute progressive renal failure with proteinuria

and anemia include anti-neutrophil cytoplasmic autoantibodies (ANCA) and antiglomerular basement membrane (GBM) glomerulonephritis [33].

Another remote cause for hematuria is development of a pseudoaneurysm in renal transplant recipient. A pseudoaneurysm is defined as arterial dilation accompanied with disruption of the one or more layers of the arterial wall. This lesion may be present at the site of puncture as a complication of procedures like arterial catheterization or as a complication of percutaneous nephrolithotomy (PCN). This procedure is done in a native or a transplanted kidney following a urinary tract obstruction and an infected hydronephrosis. Other reasons for doing a PCN include: urinary leakage, to remove calculi or a foreign body, chemotherapy and for urinary diversion due to hemorrhagic cystitis [34].

In our pilot study we observed hematuria in 35 (11.2%) patients. Of these 15 (4.8%) of a total 43 patients with active antibody mediated rejection (AMR) presented with hematuria, i.e. 34.8% patients with active AMR showed RBCs in their urine. Of 15 (4.8%) patients having recurrent glomerulonephritis, 9 (60%) presented with hematuria, five had IgA nephropathy and two each of C3 glomerulopathy and systemic lupus nephritis. None of the patients with acute tubular injury or chronic rejections or cellular rejection pr with patients with biopsy reported as unremarkable presented with hematuria. Thus, hematuria if present does indicate a disease process of graft.

#### **5.5 Urinary tract infection (UTI)**

Patients undergoing renal transplant have a suppressed immune response and hence have poor resistance to infection. Thus, infections in these group of patients is quite a common leading to morbidity and mortality post-transplantation [35]. Infections are the second most common cause for causing death in patients with renal transplant. The most common cause for predisposition of these patients to infection is that they are immunocompromised. Infection of the urinary tract is the most common infection affecting these subsets of patients, with an estimated incidence between 10 and 98% and is implicated for a longer hospital stay as well as increased health care cost [36–38].

Urine examination plays an indispensable role in diagnosis of urinary tract infection. Significant quantitative bacterial count (of ≥105 CFU/mL) in an appropriately collected urine sample aids the diagnosis of UTI in patients showing signs and symptoms of urinary tract infection [37]. Urinalysis in adjunct with urine culture studies are essential in determination of the causative organism of pyuria [39]. Presence of leucocytes in urine is an indicator of acute pyelonephritis and urinary tract infection [36, 37].

UTI can have enormous consequences on the lives of kidney recipients. For instance, it is the most common source of bloodstream infection among recipients, especially when it occurs during the first three months after transplantation [40]. Evaluations of UTI effects on renal parenchyma have shown how infections of the urinary system may result in prolonged inflammation and potential renal scarring [40, 41], which can lead to impaired renal function [42].

#### **6. Role of novel biomarkers in renal transplant recipients**

With advances in the field of renal transplantation, newer modalities for monitoring graft function have been developed. Determination of novel biomarkers in urine,

#### *Role of Urine Examination in Renal Transplant Recipients DOI: http://dx.doi.org/10.5772/intechopen.112967*

plasma, serum and tissue have been implicated in monitoring renal allograft function. According to WHO, a novel biomarker is defined as a "alteration occurring at cellular, biochemical or molecular level in cells, tissues or body fluid which can be measured and evaluated to indicate the normal biological or a pathogenic processes, or a pharmacological response to a therapeutic intervention [43, 44]. Serum creatinine level, is the most commonly used biochemical parameter to assess the renal allograft function, but is not an affective marker to detect early renal dysfunction. This happens as creatinine concentration in serum is greatly influenced even by various non-renal factors (factors influencing serum creatinine levels: body weight, race, age, gender, total body volume, drugs, muscle metabolism, protein intake) [4, 43]. Additionally, it is not able to predict or evaluate the progression of chronic injury and making it a non-specific or non-predictive marker for graft dysfunction. Alternatively, this makes the histological examination through renal allograft biopsy the gold standard to determine the immunological or non-immunological cause for graft dysfunction [4, 11]. Therefore, these biomarkers, can be used for diagnosis of patients with a disease or an abnormal organ function and also to know the severity and prognosis of a disease, as well as monitor response to a medical procedure [4]. Thus, it is predicted that estimation of these novel biomarkers could possibly help in early recognition of allograft disease as well as help in monitoring disease activity. In addition to this, it is predicted that the novel marker estimation would optimizing the need for an invasive biopsy [45–47].

However, biopsy being an invasive procedure, may not be straightforward to perform and can be complicated by major bleeding. Other drawbacks associated are: risk of potential sampling errors, the inter-observer variability in assigning Banff scores and associated cost of the procedure. Hence it is not only impractical, it is also cumbersome and economically not feasible to monitor graft function by renal biopsy. Urine, on the other hand are readily available and direct product of the allograft and have minimal influence from systemic inflammation, making it a more desirable source for biomarkers [48].

An ideal biomarker is supposed to have certain characteristics. These include readily availability, accuracy, low cost, should be easy to standardized, produce repeatable results and be non-invasive. Overall such a biomarker should be useful to reduce the necessity for performing a renal allograft biopsy and help the clinician for early management [43, 44, 48].

#### **6.1 Classification of novel biomarkers for renal allografts**

Biomarkers used to monitor renal allografts can be grouped under two broad headings [44]:


#### **6.2 Neutrophil gelatinase-associated lipocalin (NGAL) (Aka: uterocalin/ lipocalin-2, 24p3/siderocalin)**

This molecule is a member of lipocalin superfamily with molecular weight of is a 21 kD [49]. NGAL is secreted by neutrophils, acting as an acute-phase proteins [44]. First discovered as a complex protein with human neutrophil gelatinase in 1993 [49]. NGAL molecule is found in 3 isoforms: monomeric (25 kDa), dimeric (45 kDa), and as heterodimeric (135 kDa—complexed with gelatinase) [49]. The gene for this protein is located on chromosome 9 and this molecule is expressed in renal, liver, endothelial, smooth muscle cells, neurons, and cells of immune system (macrophages and dendritic cells) [50–52]. NGAL molecule expresses its action via a primary ligand, siderophore and metalloproteinase 9 (MMP-9) and is present in plasma as well as urine [4, 49]. Why is NGAL considered to be a biomarker of choice? The reason is that this biomarker is quite efficient and accurate in detecting kidney injury, very early in the post-transplant period. It is observed that there occurs a rapid rise of NGAL in urine, which is detectable even within few hours after the initial insult, whereas rise in serum creatinine occurs hours later [53]. Following AKI, the glomerular filtration rate (GFR) is also reduced which in turn causes the levels of NGAL to rise. A study showed that in patients with acute kidney injury, the levels of NGAL in blood and urine increase by 300-fold (0.1–30 μg/ml) and 1000-fold (0.04–40 mg/ml), respectively [52]. In severe cases of acute tubular injury large quantities of NGAL is excreted into urine, reaching almost up to 1000-fold. This happens due to induction of NGAL mRNA and protein in the renal epithelium as this molecule is expressed in the renal epithelium. Many studies have postulated that patients with higher urinary NGAL values in the early posttransplant phases are more prone to develop delayed graft dysfunction [53]. It has been observed that increase in serum creatinine happens several hours after renal cell destruction, but increase in urine/blood levels of NGAL can be

observed as early as two hours of inception of injury. Thus, it is suggested that NGAL can be used to assess transplant status as early as a few hours post-transplantation [4].

#### **6.3 Kidney injury molecule: 1 (KIM-1)**

This protein is a type-1 transmembrane glycoprotein. KIM-1 comprises of two domains, viz.: six-cysteine immunoglobulin-like domain and a mucin domain (extracellular) [54]. KIM-1 also known as HAVCR/TIM-1 is a protein of 104 kD and the gene for this protein is located on chromosome 5q33.2 [4, 55]. KIM-1 (designated as Kim-1 in rodents, KIM-1 in humans) mRNA was identified using techniques of representational difference analysis (which is a PCR-based technique). This technique, which was carried out to find genes, the expression of which was found to be markedly upregulated 24–48 hours after ischaemia in the rat [56]. KIM-1 is expressed in the kidney, liver, and spleen and uninjured kidney tissue. Urine expresses very low or undetectable levels of KIM-1. Studies have shown that KIM-1 plays different roles via various molecular targets in immune diseases and kidney injury. This molecule is expressed on the apical membrane surface of proximal tubular epithelial cells of the kidney (in the S3 segment) and readily responds to hypoxia and renal tubular injury. The extracellular domain of KIM-1 molecule is a quantitative marker of kidney injury and is detached by metalloproteinases and then secreted into the urine. KIM-1 is also an important marker for kidney transplant rejection [52–55]. Various studies including one by Jin et al. reported that serum KIM-1 might be a marker for the prediction of early kidney transplant rejection. They also predicted that this molecule could possibly be helpful in monitoring renal graft function in transplant recipients, and thus might contribute in early diagnosis of organ rejection [57].

#### **6.4 C-X-C motif chemokine 10 (CXCL-10)**

This molecule is an interferon-γ-inducible protein-10 (IP-10), a chemokine belonging to the CXC subfamily. This molecule consists of two cysteines that are located at the N-terminus. These two cysteines are separated by a single amino acid which can be variable [58]. The gene for this protein is located on chromosome 4. This chemokine is excreted from all the leukocytes, viz. neutrophils, eosinophils, monocytes and epithelial, endothelial, as well as stromal cells and keratinocytes. The chemokine is secreted as a response to several proinflammatory factors, like interferon-γ (IFN-γ) [58, 59]. CXCL-10 is secreted by leukocytes in the transplanted kidney and is a marker for inflammation. According to the observations of Elkman et al. CXCL9 and CXCL10, which are induced by IFNγ are supposedly to be the most studied as well as promising protein biomarkers for predicting acute renal rejection. Both CXCL9 and CXCL10 bind with CXCR3, that are expressed on activated T-cell which in turn recruit T-cells to the inflammatory site [45]. Schaub et al. demonstrated that the sensitivity and specificity of urinary CXCL-10 (uCXCL-10) exceeded those of serum creatinine levels. Various studies have been performed to determine the role of CXCL10 molecule in allogenic kidney transplant rejection. Study by Ciftci et al. which was performed on living donor related transplant recipients to assess the efficacy of CXCL10, showed that urine levels of CXCL-10 correlates well with serum creatinine level is patients having acute cellular rejection [60–62]. On the other hand, Rabant et al. studied 244 renal allotransplant recipients and monitored urinary CXCL-10 and serum creatinine levels. They further determined the ratio of CXCL10 and serum creatinine and proposed that the ratio can effectively determine the risk of antibody-dependent transplant rejection [63].

Blydt-Hansen et al. also reported similar observation for the CXCL-10 to creatinine ratio in pediatric renal transplant recipients and concluded CXCL-10 to be a promising biomarker of acute cellular rejection [64]. Matz et al. in his study reported that CXCL-10 chemokine levels may predict the development of acute cell-type rejection [65]. Watson et al. demonstrated that high pretransplant serum CXCL-10 levels may indicate a high risk of severe rejection and transplant failure and it would be appropriate to determine the CXCL-10 levels pre-transplantation [66]. Jackson et al. found that urine CXCL-10 levels can increase in acute transplant rejection as well as in patients suffering from polyoma virus nephropathy, however this chemokine cannot be used to differentiate between these two conditions [67].

#### **6.5 Calreticulin (CRT)**

CRT is a major calcium 2+ (Ca2+) binding (storage) protein. This protein is present in the lumen of the endoplasmic reticulum with a molecular weight of 46 kDa, having 400 amino acid residues. This protein is basically a major Ca2+ binding chaperon. Calreticulin has three distinct structural domains: the amino-terminal N-domain, middle P-domain, and the terminal carboxyl-C-domain along with a cleavable amino acid signal sequence. This amino acid signal sequence is present at the beginning of the N-terminal, which helps in directing CRT to the endoplasmic reticulum. The C-terminal functions for ER retention/retrieval signal. Two main functions have been implicated to this protein in the ER: One as a chaperon and other as a Ca2+ binding and storage protein. It can be identified at several other sub-cellular locations like cell surface, cytoplasm, and the extracellular matrix [68].

#### **6.6 Cystatin C (CysC)**

CysC is an endogenous proteinase inhibitor with a molecular weight of ~13.4 kD. This molecule is a member of cystatin superfamily of cysteine protease inhibitors. The main function of the protein is to inhibit cathepsins, namely cathepsin L, B, and H [69, 70]. CysC is composed of polypeptide chain having 120 amino acids and the chromosome 20 harbors the gene for this protein [71]. CysC has a role in intracellular catabolism of proteins and peptides. Another advantage of this protein is that concentration of CysC does not depend on factors like gender, age, or muscle mass, making it more suitable to determine the dynamics of GFR changes as compared to serum creatinine [72]. Krishnamurthy et al. concluded CysC as an additional diagnostic parameter in assessing the function of a transplanted organ, which additionally might be helpful and serve to tailor immunosuppressive treatment [73]. Changes in the glomerular filtration rate secondary to a deteriorating transplant function and thus an increased risk of rejection, can be detected by the determination of cystatin C according to, according to Taghizadeh Afshari et al. Study by A. Taghizadeh-Afshari showed that at 14 days post-transplant, levels of CysC exceeds the sensitivity and specificity of serum creatinine [74]. Similar observations were also made by Le Bricon et al. According to him CysC is a more accurate marker than serum creatinine. He additionally postulated role of Cystatin C in assessing the toxic effects of treatment [75].

#### **6.7 Osteopontin (OPN):0020**

Osteopontin, also known as bone sialoprotein 1 (BSP-1) or secreted phosphoprotein 1 (SPP1) and also as early T-lymphocyte activation-1 (ETA-1). This protein is

*Role of Urine Examination in Renal Transplant Recipients DOI: http://dx.doi.org/10.5772/intechopen.112967*

an extracellular matrix protein with a molecular weight of approximately 35 kD. It is composed of a polypeptide chain comprising of 314 amino acids. The polypeptide chain contains sequence of arginine-glycine-asparagine binding integrin [76–78]. This molecule is encoded by a single-copy gene which is mapped on the human chromosome 4 (4q13). This molecule is expressed on intestinal epithelial cells, bone, kidney, and immune cells, such as macrophages, dendritic cells, and the T lymphocytes [79, 80]. The serum osteopontin concentration in a normal individual is estimated to be around 23.56 ng/ml [80]. Osteopontin, in kidney, is produced at distal part of nephron. The function of this molecule is implicated in formation of renal vessels [81, 82].

#### **6.8 Clusterin (CLU)**

CLU is also called as apolipoprotein J (CLU). It is a glycosylated protein and is composed of two chains, the α-chain and β-chain. These both are linked via disulfide bonds and in human body it is present in two isoforms – secretory type and nuclear type. The mass of the secretory type is 80 kD, and is implicated in removing residues formed after apoptosis. The nuclear type isoform is 50 kD and has its role in DNA repair. The gene encoding for this protein is located on chromosome 8. Clusterin molecule has a role in apoptosis as well as in antiapoptotic pathway. CLU in human body is present in various organs, including kidney and is also detected in all biological fluids. The physiological concentrations of CLU in serum range from 35 to 105 μg/ ml. In kidney, this molecule is present in the tubules and has numerous antiapoptotic functions, by mediating cell protection, recycling of lipids, attachment and aggregation of cells. Although the function or utility of CLU in renal transplant rejection is yet to be analyzed [83–85].

#### **7. Conclusion**

Multiple causes can affect the functioning of the renal allograft, and there are multiple modalities that are recommended in evaluation of the renal transplant. In the present era where most of the investigations fall under the category of molecular tests and genetics, immunohistochemistry, cytogenetics etc., urine examination still plays an indispensable role in management of the renal allograft. Overall certain parameters like urine osmolality, proteinuria, hematuria and urine microscopy along with the newer molecules (biomarkers) are a hit and help in monitoring of the renal allograft.

#### **Author details**

Lovelesh K. Nigam Laboratory Medicine, Department of Pathology, Transfusion Services and Immunohematology, G.R. Doshi and K.M. Mehta Institute of Kidney Diseases and Research Centre and Dr. H.L. Trivedi Institute of Transplantation Sciences, Ahmedabad, India

\*Address all correspondence to: drloveleshnigam@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Role of Urine Examination in Renal Transplant Recipients DOI: http://dx.doi.org/10.5772/intechopen.112967*

#### **References**

[1] Garcia GG, Harden P, Chapman J. The global role of kidney transplantation. Kidney & Blood Pressure Research. 2012;**35**:299-304. DOI: 10.1159/000337044

[2] Abecassis M, Bartlett ST, Collins AJ, Davis CL, Delmonico FL, Friedewald JJ, et al. Kidney transplantation as primary therapy for end-stage renal disease: A National Kidney Foundation/kidney disease outcomes quality initiative (NKF/ KDOQITM) conference. Clinical Journal of the American Society of Nephrology. 2008;**3**(2):471-480. DOI: 10.2215/ CJN.05021107

[3] Shroff S. Current trends in kidney transplantation in India. Indian Journal of Urology. 2016;**32**(3):173-174. DOI: 10.4103/0970-1591.185092

[4] Rogulska K, Wojciechowska-Koszko I, Dołęgowska B, Kwiatkowska E, Roszkowska P, et al. The Most promising biomarkers of allogeneic kidney transplant rejection. Journal of Immunology Research; 2022. 18 pages. Article ID 6572338. DOI: 10.1155/ 2022/6572338

[5] Armstrong JA. Urinalysis in Western culture: A brief history. Kidney International. 2007;**71**:384-387

[6] Hashmi P, Ho C, Morgan S, Stephenson JR. Routine urinanalysis in renal transplant patients. Journal of Clinical Pathology. 1995;**48**:383-384

[7] Jenni F, Riethmüller S, Wüthrich RP. Significance of urine diagnostic tests after renal transplantation. Kidney & Blood Pressure Research. 2013;**37**:116- 123. DOI: 10.1159/000350065

[8] Kidney Disease: Improving Global Outcomes (KDIGO) Transplant Work Group. KDIGO clinical practice guideline for the care of kidney transplant recipients. American Journal of Transplantation. 2009;**9**(Suppl. 3):S1-S155

[9] Kunthara MG, Sahay M, Hussain HI, Ismal K, Vali PS, Kavadi A, et al. Posttransplant renal allograft dysfunction – A retrospective observational study. Indian Journal of Transplantation. 2021;**15**:232-240

[10] Goldberg RJ, Weng FL, Kandula P. Acute and chronic allograft dysfunction in kidney transplant recipients. Medical Clinics of North America. May 2016;**100**(3):487-503. DOI: 10.1016/j. mcna.2016.01.002. PMID: 27095641

[11] Mazloum M, Jouffroy J, Brazier F, Legendre C, Neuraz A, Garcelon N, et al. Osmoregulation performance and kidney transplant outcome. Journal of the American Society of Nephrology. 2019;**30**(7):1282-1293. DOI: 10.1681/ ASN.2018121269

[12] Zimmermann-Spinnler M. Urinlabor. Liestal CH: Medical Laboratory Consulting AG; 1991. pp. S41-S44

[13] Ondiej OS, Voska VL, Yladimir AJ, et al. Early morning urine osmolality in patients with chronic allograft nephropathy. Transplant International. 2004;**17**:270-271. DOI: 10.1007/ s00147-004-0707-6

[14] Amer H, Filder ME, Myslak M, Morales P, Kremers WK, Larson TS, et al. Proteinuria after kidney transplantation, relationship to allograft histology and survival. American Journal of Transplantation. 2007;**7**(12):2748-2756. DOI: 10.1111/j.1600-6143.2007.02006.x

[15] Stevens LA, Coresh J, Greene T, et al. Assessing kidney function–measured

and estimated glomerular filtration rate. The New England Journal of Medicine. 2006;**354**:2473-2483

[16] Knoll GA. Proteinuria in kidney transplant recipients: Prevalence, prognosis, and evidence-based management. American Journal of Kidney Diseases. 2009;**54**(6):1131-1144

[17] Suárez Fernández ML, G-Cosío F. Causes and consequences of proteinuria following kidney transplantation. Nefrología. 2011;**31**(4):404-414. English, Spanish. DOI: 10.3265/Nefrologia. pre2011.May.10972

[18] Fernández-Fresnedo G, Plaza J, Sánchez-Plumed J, Sanz-Guajardo A, Palomar-Fontanet R, Arias M. Proteinuria: A new marker of long-term graft and patient survival in kidney transplantation. Nephrology, Dialysis, Transplantation. 2004;**19**:iii47-iii51

[19] Rangan GK. Sirolimus-associated proteinuria and renal dysfunction. Drug Safety. 2006;**29**(12):1153-1161. DOI: 10.2165/00002018-200629120-00006

[20] Oblak M, Mlinšek G, Kojc N, Frelih M, Buturović-Ponikvar J, Arnol M. Spot urine protein excretion in the first year following kidney transplantation associates with allograft rejection phenotype at 1-year surveillance biopsies: An observational National-Cohort Study. Frontiers in Medicine (Lausanne). 2021;**8**:781195. DOI: 10.3389/fmed.2021.781195

[21] Khaled SM, Knoll Greg A. Posttransplantation proteinuria: An approach to diagnosis and management. Clinical Journal of the American Society of Nephrology. 2011;**6**(7):1786-1793. DOI: 10.2215/CJN.01310211

[22] Yang F, Shi JS, Gong SW, Xu XD, Le WB. An equation to estimate 24-hour total urine protein excretion rate in patients who underwent urine protein testing. BMC Nephrology. 2022;**23**(1):49. DOI: 10.1186/s12882-022-02673-2

[23] Xia M, Yang H, Tong X, Xie H, Cui F, Shuang W. Risk factors for new-onset diabetes mellitus after kidney transplantation: A systematic review and meta-analysis. Journal of Diabetes Investigation. 2021;**12**(1):109-122. DOI: 10.1111/jdi.13317

[24] Saleem MO, Hamawy K. Hematuria. [updated August 8, 2022]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Available from: https://www.ncbi.nlm.nih.gov/ books/NBK534213/

[25] Wang Z, Vathsala A, Tiong HY. Haematuria in postrenal transplant patients. BioMed Research International. 2015;**2015**:292034. DOI: 10.1155/2015/292034

[26] Trivedi HS, Lal SM, Gupta N, Ross R Jr. ATGAM associated coagulopathy in renal transplant patients: A report of two unusual cases. The International Journal of Artificial Organs. 1996;**19**(8):448-450

[27] Rivera-Sanchez R, Delgado-Ochoa D, Flores-Paz RR. Prospective study of urinary tract infection surveillance after kidney transplantation. BMC Infectious Diseases. 2010;**10**, Article 245. DOI: 10.1186/1471-2334-10-245

[28] Manikandan R, Kumar S, Dorairajan LN. Hemorrhagic cystitis: A challenge to the urologist. Indian Journal of Urology. 2010;**26**(2):159-166. DOI: 10.4103/0970-1591.65380

[29] Yagisawa T, Nakada T, Takahashi K, Toma H, Ota K, Yaguchi H. Acute Hemorrhagic cystitis caused by adenovirus after kidney transplantation. Urologia

*Role of Urine Examination in Renal Transplant Recipients DOI: http://dx.doi.org/10.5772/intechopen.112967*

Internationalis. 1995;**54**:142-146. DOI: 10.1159/000282708

[30] Schwarz A, Vatandaslar S, Merkel S, Haller H. Renal cell carcinoma in transplant recipients with acquired cystic kidney disease. Clinical Journal of the American Society of Nephrology. 2007;**2**(4):750-756

[31] Larcom RC Jr, Carter GH. Erythrocytes in urinary sediment: Identification and normal limits. With a note on the nature of granular casts. The Journal of Laboratory and Clinical Medicine. 1948;**33**(7):875-880

[32] Newman LB, Anderson EE, Schulman CC. Renal allograft rejection presenting with gross hematuria: Case report. Acta Urologica Belgica. 1972;**40**(4):825-829

[33] Nakamura T, Shirouzu T. Antibody-mediated rejection and recurrent primary disease: Two Main obstacles in abdominal kidney, liver, and pancreas transplants. Journal of Clinical Medicine. 2021;**10**(22):5417. DOI: 10.3390/jcm10225417

[34] Young M, Leslie SW. Percutaneous nephrostomy. [updated 2022 Nov 28]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Available from: https://www.ncbi.nlm. nih.gov/books/NBK493205/

[35] Singh N, Limaye AP. Infections in solid-organ transplant recipients. In: Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. 2015. pp. 3440-3452. DOI: 10.1016/B978-1-4557-4801-3.00313-1. PMCID: PMC7151835

[36] Anastasopoulos NA, Duni A, Peschos D, Agnantis N, Dounousi E. The Spectrum of infectious diseases in kidney transplantation: A review

of the classification, pathogens and clinical manifestations. Infectious Diseases in Kidney Transplantation. 2015;**29**:415-422

[37] Nigam LA, Vanikar AV, Patel RD, Kanodia KV, Suthar KS. Urinary Tract Infection in Renal Allograft Recipents. In: Tomas J, Daca A, Dębska-Ślizień MA, editors. London, UK: IntechOpen; [Internet]. 2018. DOI: 10.5772/intechopen.77171

[38] Olenski S, Scuderi C, Choo A, Bhagat Singh AK, Way M, Jeyaseelan L, et al. Urinary tract infections in renal transplant recipients at a quaternary care Centre in Australia. BMC Nephrology. 2019;**20**(1):479. DOI: 10.1186/ s12882-019-1666-6

[39] Goldman JD, Julian K. Urinary tract infections in solid organ transplant recipients: Guidelines from the American Society of Transplantation infectious diseases Community of Practice. Clinical Transplantation. 2019;**33**(9):e13507. DOI: 10.1111/ctr.13507

[40] Memikoglu KO, Keven K, Sengul S, et al. Urinary tract infections following renal transplantation: A single-center experience. Transplantation Proceedings. 2007;**39**(10):3131-3347

[41] Ramsey DE, Finch WT, Birtch AG. Urinary tract infections in kidney transplant recipients. Archives of Surgery. 1979;**114**(9):1022-1025

[42] Ariza-Heredia EJ, Beam EN, Lesnick TG, Kremers WK, Cosio FG, Razonable RR. Urinary tract infections in kidney transplant recipients: Role of gender, urologic abnormalities, and antimicrobial prophylaxis. Annals of Transplantation. 2013;**18**:195-204. DOI: 10.12659/AOT.883901

[43] Wang H, Lin ZT, Yuan Y, Wu T. Urine biomarkers in renal allograft. Journal

of Translational Internal Medicine. 2016;**4**(3):109-113. DOI: 10.1515/ jtim-2016-0032

[44] Swanson KJ, Aziz F, Garg N, Mohamed M, Mandelbrot D, Djamali A, et al. Role of novel biomarkers in kidney transplantation. World Journal of Transplantation. 2020;**10**(9):230-255 Available from: https://www.wjgnet. com/2220-3230/full/v10/i9/230.htm. DOI: 10.5500/wjt.v10.i9.230

[45] Eikmans M, Gielis EM, Ledeganck KJ, Yang J, Abramowicz D, Claas FFJ. Noninvasive biomarkers of acute rejection in kidney transplantation: Novel targets and strategies. Frontiers in Medicine (Lausanne). January 2019;**5**(8):358. DOI: 10.3389/fmed.2018.00358. PMID: 30671435; PMCID: PMC6331461

[46] Naesens M, Anglicheau D. Precision transplant medicine: Biomarkers to the rescue. Journal of the American Society of Nephrology. 2018;**29**:24-34

[47] Wiebe C, Ho J, Gibson IW, Rush DN, Nickerson PW. Carpe diem-time to transition from empiric to precision medicine in kidney transplantation. American Journal of Transplantation. 2018;**18**:1615-1625

[48] Guzzi F, Cirillo L, Buti E, Becherucci F, Errichiello C, Roperto RM, et al. Urinary biomarkers for diagnosis and prediction of acute kidney allograft rejection: A systematic review. International Journal of Molecular Sciences. 2020;**21**(18):6889. DOI: 10.3390/ijms21186889

[49] Ning M, Mao X, Niu Y, Tang B, Shen H. Usefulness and limitations of neutrophil gelatinase-associated lipocalin in the assessment of kidney disease. The Journal of Laboratory and Precision Medicine (JLPM). 2018;**3**(1). DOI: 10.21037/jlpm.20

[50] Al-Refai AA, Tayel SI, Ragheb A, et al. Urinary neutrophil gelatinase associated lipocalin as a marker of tubular damage in type 2 diabetic patients with and without albuminuria. Open Journal of Nephrology. 2014;**4**(1):37-46

[51] Szumilas D, Wojnar J, Chudek J. Neutrophil gelatinase-associated lipocalin as a marker of acute renal failure in cancer patients treated with cisplatin. Nowotwory. Journal of Oncology. 2016;**66**(2):160-166

[52] Beker B, Corleto M, Fieiras C, Musso C. Novel acute kidney injury biomarkers: Their characteristics utility and concerns. International Urology and Nephrology. 2018;**50**(4):705-713

[53] Cappuccilli M, Capelli I, Comai G, Cianciolo G, La Manna G. Neutrophil gelatinase-associated Lipocalin as a biomarker of allograft function after renal transplantation: Evaluation of the current status and future insights. Artificial Organs. 2018;**42**(1):8-14. DOI: 10.1111/aor.13039

[54] Bailly V, Zhang Z, Meier W, Cate R, Sanicola M, Bonventre JV. Shedding of kidney injury molecule-1, a putative adhesion protein involved in renal regeneration. Journal of Biological Chemistry. 2002;**277**(42): 39739-39748

[55] Medić B, Rovčanin B, Basta Jovanović G, Radojević-Škodrić S, Prostran M. Kidney injury molecule-1 and cardiovascular diseases: From basic science to clinical practice. BioMed Research International. 2015;**2015**:854070. . DOI: 10.1155/2015/854070. PMID: 26697493; PMCID: PMC4677159

[56] Bonventre JV. Kidney injury molecule-1 (KIM-1): A urinary

*Role of Urine Examination in Renal Transplant Recipients DOI: http://dx.doi.org/10.5772/intechopen.112967*

biomarker and much more. Nephrology, Dialysis, Transplantation. 2009;**24**:3265- 3268. DOI: 10.1093/ndt/gfp010

[57] Jin ZK, Tian P, Wang X, et al. Kidney injury molecule-1 and osteopontin: New markers for prediction of early kidney transplant rejection. Molecular Immunology. 2013;**54**(3-4):457-464

[58] Neville LF, Mathiak G, Bagasra O. The immunobiology of interferon-gamma inducible protein 10 KD (IP-10): A novel, pleiotropic member of the C-X-C chemokine superfamily. Cytokine & Growth Factor Reviews. 1997;**8**(3):207-219

[59] Zlotnik A, Yoshie O. Chemokines: A new classification system and their role in immunity. Immunity. 2000;**12**(2):121-127

[60] Liu M, Guo S, Hibbert J, et al. CXCL10/IP-10 in infectious diseases pathogenesis and potential therapeutic implications. Cytokine & Growth Factor Reviews. 2011;**22**(3):121-130

[61] Schaub S, Nickerson P, Rush D, et al. Urinary CXCL9 and CXCL10 levels correlate with the extent of subclinical tubulitis. American Journal of Transplantation. 2009;**9**(6):1347-1353

[62] Ciftci HS, Tefik T, Savran MK, et al. Urinary CXCL9 and CXCL10 levels and acute renal graft rejection. International Journal of Organ Transplantation Medicine. 2019;**10**(2):53-63

[63] Rabant M, Amrouche L, Lebreton X, et al. Urinary C-X-C motif chemokine 10 independently improves the noninvasive diagnosis of antibody-mediated kidney allograft rejection. Journal of the American Society of Nephrology. 2015;**26**(11):2840-2851

[64] Blydt-Hansen TD, Gibson IW, Gao A, Dufault B, Ho J. Elevated urinary CXCL10-to-creatinine ratio is associated with subclinical and clinical rejection in pediatric renal transplantation. Transplantation. 2015;**99**(4):797-804

[65] Matz M, Beyer J, Wunsch D, et al. Early post-transplant urinary IP-10 expression after kidney transplantation is predictive of short- and long-term graft function. Kidney International. 2006;**69**(9):1683-1690

[66] Watson D, Yang JYC, Sarwal RD, et al. A novel multibiomarker assay for non-invasive quantitative monitoring of kidney injury. Journal of Clinical Medicine. 2019;**8**(4):499

[67] Jackson JA, Kim E, Begley B, et al. Urinary chemokines CXCL9 and CXCL10 are noninvasive markers of renal allograft rejection and BK viral infection. American Journal of Transplantation. 2011;**11**(10):2228-2234

[68] Gawish RIAR, El Aggan HAM, Mahmoud SAH, et al. A novel biomarker of chronic allograft dysfunction in renal transplant recipients (serum calreticulin and CD47). The Egyptian Journal of Internal Medicine. 2020;**32**:19. DOI: 10.1186/s43162-020-00018-9

[69] Murty MSN, Sharma UK, Pandey VB, Kankare SB. Serum cystatin C as a marker of renal function in detection of early acute kidney injury. Indian Journal of Nephrology. 2013;**23**(3):180-183

[70] Cimerman N, Prebanda MT, Turk B, Popovič T, Dolenc I, Turk V. Interaction of cystatin C variants with papain and human cathepsins B, H and L. Journal of Enzyme Inhibition. 1999;**14**(2):167-174

[71] Mareš J, Stejskal D, Vavroušková J, Urbánek K, Herzig R, Hluštík P. Use of cystatin C determination in clinical diagnostics. Biomed Papers. 2003;**147**(2):177-180

[72] Chew JS, Saleem M, Florkowski CM, George PM. Cystatin C–a paradigm of evidence based laboratory medicine. The Clinical Biochemist Reviews. 2008;**29**(2):47

[73] Krishnamurthy N, Arumugasamy K, Anand U, Anand CV, Aruna V, Venu G. Serum cystatin C levels in renal transplant recipients. Indian Journal of Clinical Biochemistry. 2011;**26**(2):120-124

[74] Taghizadeh-Afshari A, Mohammadi-Fallah M, Alizadeh M, et al. Serum cystatin C versus creatinine in the assessment of allograft function in early periods of kidney transplantation. Journal of Renal Injury Prevention. 2017;**7**(1):11-15

[75] Le Bricon T, Thervet E, Benlakehal M, Bousquet B, Legendre C, Erlich D. Changes in plasma cystatin C 16 journal of immunology research after renal transplantation and acute rejection in adults. Clinical Chemistry. 1999;**45**(12):2243-2249

[76] Wai PY, Kuo PC. The role of osteopontin in tumor metastasis. Journal of Surgical Research. 2004;**121**(2):228-241

[77] Christensen B, Nielsen MS, Haselmann KF, Petersen TE, Sørensen ES. Post-translationally modified residues of native human osteopontin are located in clusters: Identification of 36 phosphorylation and five O-glycosylation sites and their biological implications. Biochemical Journal. 2005;**390**(Part 1):285-292

[78] Young MF, Kerr JM, Termine JD, et al. CDNA cloning, MRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN). Genomics. 1990;**7**(4):491-502

[79] Brown LF, Berse B, Van de Water L, et al. Expression and distribution of osteopontin in human tissues: Widespread association with luminal epithelial surfaces. Molecular Biology of the Cell. 1992;**3**(10):1169-1180

[80] Reza S, Shaukat A, Arain TM, Riaz QS, Mahmud M. Expression of osteopontin in patients with thyroid dysfunction. PLoS One. 2013;**8**(2):e56533. DOI: 10.1371/journal. pone.0056533

[81] Hudkins KL, Giachelli CM, Cui Y, Couser WG, Johnson RJ, Alpers CE. Osteopontin expression in fetal and mature human kidney. Journal of the American Society of Nephrology. 1999;**10**(3):444-457

[82] Rogers SA, Padanilam BJ, Hruska KA, Giachelli CM, Hammerman MR. Metanephric osteopontin regulates nephrogenesis in vitro. American Journal of Physiology Renal Physiology. 1997;**272**(4):469-476

[83] De Silva HV, Harmony JAK, Stuart WD, Gil CM, Robbins J. Apolipoprotein J: Structure and tissue distribution. Biochemistry. 1990;**29**(22):5380-5389

[84] Pajak B, Orzechowski A. Clusterin: The missing link in the calciumdependent resistance of cancer cells to apoptogenic stimuli. Postepy Higieny I Medycyny Doswiadczalnej (Online). 2006;**60**:45-51

[85] Jenne DE, Tschopp J. Clusterin: The intriguing guises of a widely expressed glycoprotein. Trends in Biochemical Sciences. 1992;**17**(4):154-159

#### **Chapter 5**

### Application of Urine Metabolomics as a Marker in Health and Disease

*Abraham Joseph Pellissery, Poonam Gopika Vinayamohan, Leya Susan Viju, Divya Joseph and Kumar Venkitanarayanan*

#### **Abstract**

Advances in metabolomics research have yielded an avenue for utilizing this laboratory-based modality as a platform for clinical diagnosis, identification of novel biomarkers, and longitudinally monitoring the health status of individuals from normal physiological and pathophysiological perspectives. This chapter provides insight on the application of urinalysis in health and disease from the standpoint of deciphering a larger span of metabolite and biomarker identification using metabolomics, specifically focusing on infectious diseases, oncology, metabolic, and inflammatory diseases in humans.

**Keywords:** urine metabolome, urinalysis, pre-analytical factors, cancer, infectious disease, inflammation, metabolic disease, renal disease

#### **1. Introduction**

Urinalysis or uroscopy is the science of disease diagnosis by means of observation and examination of the urine. Urinalysis has been considered as an adjunct of all laboratory tests applied for medical diagnosis by correlating with the symptoms exhibited by patients and is historically referenced as the first body fluid to be studied scientifically [1, 2]. Ancient medical literature from India and China have referenced the accumulation of ants and insects around sites of urination of certain individuals who had obviously suffered from diabetes. The science of uroscopy was advocated in 300 BC by Hippocrates and was considered as a popular testing modality to link his observations with the doctrine of the four humors, that is, the phlegm, blood, yellow bile, and black bile. During those times, analysis of urine color, consistency, transparency, odor, and the presence or absence of froth aided to make a general assessment of the balance of the four humors and possibly location of the disease within the body and overall prognosis [2]. Throughout the medieval and post-seventeenth century periods, medical literature had cited urinalysis as an important foundation for medical practice. Although medical practice during the mid-nineteenth century had done away with the practice of uroscopy due to the advancements in human medicine, urinalysis still prevails as an important foundation and a powerful tool for clinical diagnosis [3]. Urinalysis has been used to identify genetic diseases as well as diagnose pathophysiological processes by measuring abnormal urine constituents such as

glucose, bile pigments, white blood cells, proteins, etc. In addition, the presumptive diagnosis of diseases affecting the urogenital system can be inferred through urinalysis [3–5]. This chapter describes the applications of urine metabolomics in defining the biomarkers of health and clinical disease states.

#### **2. Metabolomics in clinical urinalysis**

Urine is an easily accessible biological fluid for noninvasive collection of large volumes with the possibility of repeat sampling at different time points, as needed for monitoring health status [6]. Traditionally, urinalysis tests conducted in diagnostic laboratories measured only one or two metabolite components (e.g., glucose, ketone bodies, etc.) at a given time [7]. Also, the abnormal urinary constituents tested via traditional methods lack specificity and are noticeable in urine samples after tissue injury. Therefore, an ideal biomarker in urine should be highly sensitive and specific and should be capable of indicating an early phase of disease progression [8].

Since the 1970s, urinary metabolome analysis has facilitated in investigation of metabolic signatures or fingerprints of urinary metabolites during the disease process in the human body [4, 8, 9]. Metabolomics is defined as the systemic identification and quantification of all metabolites in a given organism or biological sample [10]. Since metabolites generated in an organism are a result of several gene-level (host-dependent, genetic factors) and environmental-level interactions, the general metabolome, which includes the sum of all the metabolites in an organism, can serve as a critical biomarker analyte fingerprint for the health status of an individual's phenotype. Emerging technologies based on mass spectrometry (MS) and nuclear magnetic resonance spectrometry (NMR) enable the monitoring of hundreds of metabolites from tissues or body fluids. Metabolites change rapidly in response to physiological alterations, thus they act as the first-line chemical reporters of abnormal or disease phenotypes. Current advancements in high-resolution metabolomics platforms capable of detecting hundreds of low-molecular-weight metabolites in tissues and body fluids have evolved as a powerful biomarker analysis tool. The datasets generated from clinical trials and research studies curate for clinical databases that support diagnosis and therapeutic assessment of several disease states [7]. However, these datasets require standardization and validation to create a clinical reference guide that is applicable for any biofluid, as well as FDA approval for utilizing this platform for clinical diagnostic purposes [11].

#### **3. Sample collection considerations for urine metabolomics**

Urine metabolome has aided in the examination of metabolic consequence correlating to disease, nutritional status, and environmental toxicity since excreted urine contains endogenous and exogenous metabolites, thereby providing a key biomarker fingerprint to diagnose, monitor, or predict for any pathophysiological condition [12]. More than 3100 metabolites have been characterized in human urine, and the list is expected to increase as more and more low-concentration metabolites are being characterized [9]. With the intended application of urine samples for biomedical research or clinical diagnosis, pre-analytical factors such as collection methods and preprocessing, transport and storage, freeze-thaw cycles, and sample normalization are taken into consideration when processing metabolomic analysis [13]. Standardization

#### *Application of Urine Metabolomics as a Marker in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.109808*

of pre-analytical process is critical for minimizing inter-sample variability issues and maintaining the metabolic integrity of samples so that the metabolomic profile accurately reflects the *in vivo* biochemical status of the patient [13].

Generally, three kinds of collection modalities are employed for metabolomic studies involving urine samples: (a) first-morning void (b) spot urine samples, and (c) 24 h urine collection. The first-morning void urine samples are preferred since the overnight fast period helps to reduce the effect of any medication or the meal consumed from the previous day. Spot urine samples are the preferred sample type for dietary or pharmaceutical intervention studies and are usually collected during the daytime. However, pooled urine void samples during a 24 h duration reduce the influence of circadian cycle variation when compared to first void or spot urine samples [13].

Urine samples held at room temperature for short periods of time can lead to rapid degradation of metabolites. Considering the storage requirements of urine samples, it is optimal to freeze samples as soon as possible. Generally, while conducting clinical metabolomic studies, samples are refrigerated in autosamplers of MS and NMR instruments for time spans from hours to days. Ideally, it has been indicated by researchers that keeping samples at 4°C for 48 h or less does not significantly affect the urinary metabolome [9]. Although it would be prudent to minimize multiple freezing and thawing of urine samples, up to 9 freeze-thaw cycles are considered amenable for urine samples utilized for metabolomic studies. For long-term storage of up to 6 months or more, temperatures between −20 and −80°C are recommended. Another concern with urine collection and storage is bacterial contamination, which can be curtailed by collecting mid-stream urine and subsequently adding antibacterial agents such as sodium azide or sodium fluoride [9]. As an alternative, storage of samples at −80°C over the use of antibacterial additives can prevent the microbial metabolism of urinary metabolites, thereby rendering the urine sample suitable for downstream metabolomic applications. In addition, to reduce the metabolite transformation subsequent to sample collection, snap-freezing in liquid nitrogen aids in metabolic quenching (i.e., inactivation of enzymatic reactions), thereby obtaining an accurate picture of the metabolome at sampling time [9, 13].

The health status and fluid intake of sample submitters could also impact the solute concentration of urine. In such situations, pre-analytic normalization is essential to correct for variations in urinary constituents. Urine samples are normalized by measuring the osmolality or specific gravity, and subsequent dilution to the lowest concentration before running samples in separation and detection analytical platforms for metabolomics [9].

#### **4. Analytical techniques used in metabolomics**

Metabolomics provides a global analysis of several classes of metabolites with diverse physicochemical characteristics present in any biological sample. In general, metabolomic profiling employs two major analytical techniques for detection of metabolites, such as high-field NMR and MS. Advanced chromatographic separation techniques coupled with the aforesaid spectroscopic or spectrometric methods aid in efficiently separating complex matrices for effective detection. When comparing the detection methods, although NMR is a robust and nondestructive method, it has a lower sensitivity compared to MS. However, NMR is capable of structural elucidation of novel unknown compounds. On the other hand, MS is a very sensitive method that requires sample preparation steps coupled with suitable separation techniques to reduce ion suppression. For untargeted metabolomics, high-resolution MS instrumentation is required, whereas targeted metabolomics employ low-resolution MS platforms [5].

#### **5. Clinical research studies utilizing urine metabolomics as a diagnostic platform**

Several research studies have focused on evaluating urinary biomarkers for clinically assessing the progression of a disease or different stages of disease evolution. These biomarkers can aid in the clinical assessment of patients without apparent disease, with suspected disease, or rather the progression or remission of overt disease [14]. The following sections describe some of the clinical conditions that have been studied by researchers for potentially identifying biomarkers in urine for diseases in general, such as cancers, metabolic syndromes, and infectious and renal diseases.

#### **5.1 Cancer**

Early advances in metabolomics technology have provided insight into the metabolism of cancer by focusing on how the Warburg effect in cancerous cells uses glycolysis effectively to produce amino acids, nucleotides, and lipids necessary for tumor proliferation. Furthermore, metabolomics research has also been used to discover novel diagnostic cancer biomarkers to better understand its complex heterogeneous nature, to discover pathways involved in cancer that could be used for identification of new targets, and for monitoring metabolic biomarkers for therapeutic aspects of cancer treatment. The metabolomics approach also provides ways to personalized cancer treatments by yielding essential information regarding the cancer patient's response to medical interventions. Urine contains metabolic signatures of many biochemical pathways and thus gives way for a cohesive metabolomic approach. Techniques such as hydrophilic interaction chromatography (HILIC-LC-MS), reversed-phase ultra-performance liquid chromatography (RP-UPLC-MS), and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) are used for urine analysis [15].

Gas chromatography/mass spectrometry (GC/MS) has been recently used to explore disease biomarkers from urine or serum samples. Both primary and secondary metabolites can be analyzed as a metabolomic approach for biomarker analysis. The importance of early diagnosis is especially true in kidney cancer, especially prior to metastatic spread, and can improve survival rates from 10% to greater than 90%. The ultimate composition of molecules in the urine is the result not only of glomerular filtration but also of tubular secretion and reabsorption. Normal urine contains approximately 150 mg/24 h of protein, and compounds such as inulin (5 kDa) and lysozyme (14 kDa) are reported to appear freely in the urine. Although kidney cancer is the sixth leading cause of cancer death and represents only 3% of cancer incidence, it is a major cause of death in 11,000 patients per year in the United States. The disease is very resistant to chemotherapy, and one-third of the cases are metastatic at diagnosis. Thus when detected with symptoms, prognosis of renal cell carcinoma (RCC) is poor and once metastatic, it has only a 5% five-year survival rate. Therefore, a novel, convenient, and noninvasive approach is essential for identifying RCC at an earlier stage prior to metastasis [16]. Renal cell carcinoma entails abundant

#### *Application of Urine Metabolomics as a Marker in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.109808*

primary and secondary metabolites as potential tumor markers. The human kidney injury molecule-1 (hKIM-1), when normalized to creatinine, appears in the urine of RCC patients and disappears or decreases in concentration after nephrectomy. Hence, detection of such metabolites in urine can be crucial to cancer diagnosis. It is highlighted that metabolomics is ideally suited for such approaches and analysis of these small molecule metabolites that appear in both serum and urine can be crucial [15].

Metabolomics has also been used to investigate the urinary metabolite differences between hepatocellular carcinoma (HCC) male patients and normal male subjects. The urinary endogenous metabolome was assayed using chemical derivatization followed by GC/MS. After GC/MS analysis, 103 metabolites were detected, of which 18 metabolites were shown to be significantly different between the HCC and control groups. Subsequently, a diagnostic model was constructed with a combination of 18 marker metabolites. This noninvasive technique of identifying HCC biomarkers from urine has potential application in clinical diagnostic oncology. Overall, these findings underscore that metabolomic analysis is a potent and promising strategy for identifying novel biomarkers of HCC [17].

#### **5.2 Metabolic syndrome**

The "metabolic syndrome" (MetS) can be understood as a clustering of components that reflect overnutrition, sedentary lifestyles, and resultant excess adiposity. MetS is a cluster of different conditions and not a single disease. The prevalence of the MetS is increasing to epidemic proportions in the United States and also in developing nations. MetS is associated with doubling of incidence of cardiovascular disease risk and an increased risk for incident type 2 diabetes mellitus [18].

Accurate predictors of cardiometabolic diseases are of particular importance since the condition can be present long before the symptoms become clinically apparent. Nuclear magnetic resonance spectroscopy and gas- or liquid-chromatography coupled with MS are the major platforms applied to identify predictive biomarkers, monitoring therapeutic response as well as in basic mechanism studies of obesity, metabolic syndrome, type 2 diabetes, and cardiometabolic diseases for early diagnosis. Nicotinuric acid is correlated with cardiometabolic risk factors such as body mass index (BMI), blood pressure, HbA1c, blood lipids, and C-reactive protein, thus suggesting that it could be a potential biomarker of important features of MetS such as altered lipid metabolism and increased insulin resistance [18, 19].

Metabolic profiling of urine samples has also been used as a diagnostic tool in the predicting liver disease progression because traditional clinical chemistry tests for liver function only aid in diagnosis after substantial liver damage has occurred. With the current diagnostic methods incapable of predicting typical Jaundice syndrome (JS) in hepatic dysfunction, Wang et al. [20] conducted a study for the identification of potential biomarkers from JS disease by using a nontarget metabolomics method and testing their usefulness in human JS diagnosis. To identify the potential biomarkers, multivariate data analysis methods were utilized revealing 44 marker metabolites contributing to the complete separation of JS from healthy controls [20]. Targeted metabolite analysis revealed alterations in critical JS metabolic pathways, such as glutamate metabolism, synthesis and degradation of ketone bodies, alanine and aspartate metabolism strongly associated with JS development [20]. In another study, researchers compared the urine metabolome panel of three human subject group categories, namely individuals with nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), as well as age and sex-matched healthy controls. Urine

metabolomic analysis performed using tandem LC-MS revealed differences in 31 metabolites between NASH and NAFLD groups, including variations in nucleic acids and amino acids. Among the overlapping metabolites, it was inferred that pathways of energy and amino acid metabolism, as well as the pentose phosphate pathway, were closely associated with progression of NAFLD and NASH [21].

UHPLC-Q-TOF-MS based metabolomics approach was applied to gain understanding of the global profiling of endogenous metabolites in urine from high-fat diet-induced obese rats [22]. Integrated with multivariate analysis, metabolic variations between the obese rats and healthy rats were differentiated. Twenty potential biomarkers were identified in response to high-fat diet-induced obesity. Seventeen of them are novel potential biomarkers that are independent of the known risk indicators for obesity, except hippurate, phenylacetylglutamine (PAG), and creatinine. Using the correlation between these biomarkers, a network diagraph was generated based on search results from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Tryptophan metabolism, phenylalanine and tyrosine metabolism, and gut microbiota metabolism were found to be significantly disturbed in obese rats [22].

#### **5.3 Urinary metabolites associated with inflammatory diseases**

Immune-mediated inflammatory diseases are a group of diseases that share common molecular mechanisms and are characterized by aberrant and chronic activation of the immune system. Rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, Crohn's disease, and ulcerative colitis are the most prevalent immune-mediated inflammatory diseases. Although these diseases target different tissues and organs, they share many genetic loci, and clinically similar inflammatory diseases are known to share specific hub metabolites such as citrate. Numerous high-throughput analysis technologies are available that can generate comprehensive profiles of multiple metabolites. However, most of these techniques require invasive sampling procedures. Understanding and identifying biological markers in urine that accurately correlate with the inflammatory disease can prove vital for easy and early disease diagnosis under routine clinical settings.

A study conducted by Alonso and coworkers identified multiple urinary metabolites (citrate, alanine, methyl succinate, trigonelline, N-acetyl Amino Acids, and an unknown metabolite) that can be associated with three or more of the immunemediated inflammatory diseases [23]. Most of these urinary metabolites were found in lower concentrations in patients with inflammatory diseases compared to controls. Citrate, the strongest hub metabolite, for example, was present at lower concentrations in the urine of inflammatory bowel disease, rheumatoid arthritis, and, systemic lupus erythematosus patients [23–25]. Additionally, inflammatory diseases with similar phenotypes exhibited similar urinary metabolomes. Low levels of carnitine were identified in chronic arthritis diseases, namely rheumatoid arthritis and psoriatic arthritis. Similarly, reduced concentrations of hippurate were observed in patients with inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. In short, immune-mediated inflammatory diseases can be aggregated into three distinct clusters based on the urinary metabolite profiles: (1) psoriasis and psoriatic arthritis, (2) Crohn's disease and ulcerative colitis, and (3) rheumatoid arthritis and systemic lupus erythematosus, all sharing between 3 and 6 metabolite associations [23]. However, since the treatment strategies of Crohn's disease and ulcerative colitis are entirely different, a single test that distinguishes the two will be of utmost clinical value. In this context, researchers have identified that hippurate

#### *Application of Urine Metabolomics as a Marker in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.109808*

and 4-cresol sulfate levels were lower in patients with Crohn's disease when compared to control and ulcerative colitis patients. Although similar studies showed that urinary metabolome is useful for the differential diagnosis between ulcerative colitis and Crohn's disease, complicated nature of the disease and the confounding factors such as surgical resections, drug and dietary therapy can interfere with the metabolic changes in observational studies. Therefore, the aforesaid confounders should be considered before such analysis [24, 26]. Urinary metabolome also has the potential for predicting both the disease activity and disease recurrence (especially at the site of surgery) in patients with Crohn's disease. For example, metabolites namely citrate, hippurate and 3-hydroxyisovalerate were found in much lower levels in patients with high disease activity for Crohn's disease than in low disease activity for Crohn's disease patients [23, 27]. Higher levels of three urinary metabolites (L-3,4-dihydroxy phenylalanine, levoglucosan, ethyl malonate), and lower concentrations of propylene glycol were associated with endoscopic recurrence in Crohn's s disease in patients who have undergone ileocolonic resection [27]. Furthermore, urinary metabolites, octanoyl glucuronide, pyridoxic acid, and pantothenic acid were shown as dietary biomarkers for clinical remission in pediatric patients with inflammatory bowel disease, who had undergone either exclusive enteral nutrition or corticosteroid therapy [28].

In addition, there are distinct urinary metabolites such as phenyl acetyl glycine and tyrosine that were specific for ulcerative colitis and rheumatoid arthritis, respectively [23]. Urinary excretion of prostaglandins thromboxane synthase and prostacyclin metabolites were increased in patients with severe atherosclerosis [29]. Similarly, elevated levels of acotinic acid, isocitric acid, and citric acid were observed in the urine of osteoarthritic patients and these provide an indication of mitochondrial dysfunction leading to impaired cartilage and chondrocyte metabolism in osteoarthritis. Moreover, significant urinary metabolomic variations in histidine and histamine were observed between two different phenotypes of osteoarthritis (with and without knee effusion) [30]. The NMR-based approach also demonstrated the metabolic fingerprints of urine samples that distinguished chronic inflammatory rheumatoid diseases from healthy individuals. Several urinary metabolites (including leucine, valine, 3-hydroxyisobutyric acid, 3-hydroxyisovaleric acid, glycine, citric acid, creatinine, hippuric acid, and methylnicotinamide) were downregulated in patients with chronic inflammatory rheumatoid diseases. Some of the changes could be explained as a consequence of urinary tract infections, increased demand for muscle turnover events, or due to distal renal tubular acidosis [31]. As for pelvic inflammatory diseases, a clinical trial conducted by Zou and coworkers showed the presence of eighteen differential metabolites in the urine of rats inoculated with *Ureaplasma urealyticum* and pathogenic *Escherichia coli* to mimic multi-pathogenic infection of the upper genital tract leading to pelvic inflammation [32].

#### **5.4 Infectious diseases**

Urine metabolomics has been increasingly used for the study of biomarker discovery in infectious diseases, as it offers significant methodological advantages. The application of NMR spectroscopy metabolomics has the potential for infectious disease diagnosis since it can differentiate between various viral and bacterial infections. A specific metabolomic response comes from the host in the form of immune cells and apoptosis signaling when a pathogen causes infection [33, 34].

Urinary tract infection (UTI) is one of the most common bacterial infections in humans. Main causative organisms of UTI include *Escherichia coli, Klebsiella* 

*pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis.* Ultra-Fast Liquid Chromatography Mass Spectrometry (UFLC-MS) was used to differentiate UTI by *E. coli* in a case study involving 17 individuals. Metabolic discriminators identified were related to TCA cycle, terpenoid backbone biosynthesis, amino sugars and nucleotide sugars metabolism, arachidonic acid, and steroid hormone biosynthesis [35]. In another study, nontargeted exploratory UPLC-MS-based approach was used for the investigation of UTI-related changes in urine associated with *E. coli* infection in 117 subjects. A C-terminal glycopeptide of the human fibrinogen alpha-chain was identified as the discriminator metabolite [36]. NMRbased screening showed that *K. pneumoniae* causing UTI can metabolize glycerol to 1,3-propanediol (1,3-PD), acetate, ethanol, and succinate. The quantity of 1,3-PD was found to be proportional to the bacterial count. However, other bacteria causing UTI cannot metabolize glycerol under similar conditions [37, 38]. Urinary NMR spectroscopy of samples infected with *E. coli*, *K. pneumoniae*, *P. aeruginosa*, and *P. mirabilis* showed peaks of nonspecific metabolites such as succinate, acetate, lactate, and ethanol compared to healthy groups in a case-control study done in 617 people. Lactate metabolism, nicotinic acid, and methionine metabolism were altered in the affected individuals [39].

Pneumonia is caused by a wide range of microorganisms, including bacteria, fungi, viruses, and parasites. Conventional methods of diagnosis are time-consuming and include isolation of organisms from blood, sputum, pleural fluid, and bronchoalveolar lavage. Urinary metabolomics can be used as a tool to differentiate *Streptococcus pneumoniae* infection, responsible for community-acquired pneumonia from other infections. A study was conducted on 641 individuals, including healthy volunteers, patients with metabolic stress, fasting individuals, patients with pneumococcal pneumonia, other lung infections, and asthma or chronic obstructive pulmonary disease (COPD). NMR spectra comparison of 61 metabolites in urine from age and gender-matched *S. pneumoniae* infected and noninfected groups were performed. Among these metabolites, 6 were significantly decreased while 27 were significantly increased. Six metabolites that decreased are associated with TCA cycle intermediates (citrate, and succinate), nicotinamide metabolism (1-methylnicotinamide), food intake (levoglucosan and trigonelline), and protein catabolism (1-methylhistidine). Increased concentration was observed in amino acids (alanine, asparagine, isoleucine, leucine, lysine, serine, threonine, tryptophan, tyrosine, and valine), fatty acid oxidation (3-hydroxybutyrate, acetone, carnitine, and acetylcarnitine), inflammation (hypoxanthine and fucose), metabolites involved in glycolysis (glucose and lactate), osmolytes (*myo*-inositol and taurine), acetate, quinolinate, adipate, dimethylamine, and creatine. TCA cycle intermediates 2-oxoglutarate and fumarate also appeared to increase upon pneumococcal infection. Metabolites that were not affected by pneumococcal infection included creatinine, 3-methylhistidine, aconitate (*trans* and *cis*), metabolites related to gut microflora (3-indoxylsulfate, 4-hydroxyphenylacetate, hippurate, formate and TMAO (trimethylamine-*N*-oxide), dietary metabolites (mannitol, propylene glycol, sucrose, and tartrate) and certain amino acids (glycine, glutamine, histidine, and pyroglutamate) [38, 40]. In another study, metabolomic profiling of an independent sample set of 145 urine samples from healthy individuals or patients with various conditions showed around 86% and 94% in sensitivity and specificity, respectively, for the diagnosis of pneumococcal pneumonia [40].

Neonatal sepsis is an infection that occurs in the bloodstream of newborn infants less than 28 days old, and it can be divided into early-onset and late-onset. A case study was conducted to analyze the difference in the urinary metabolome of infected

#### *Application of Urine Metabolomics as a Marker in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.109808*

and healthy neonates [41]. Urine metabolic profiles were assessed using nontargeted NMR spectroscopy and targeted liquid chromatography-tandem mass spectrometry analysis in 16 septic neonates and 16 nonseptic ones. The metabolic profile of neonates with sepsis was found to be different compared to those without sepsis. Metabolites from energy-producing biosynthetic pathways and basic structural components of the organism showed clear separation. Elevations in urinary taurine and hypotaurine were noted in septic neonates. Depletion in glutamine levels was also seen in critically ill adults. Hypo- or hyperglycemia was also common in adults. Increased amounts of pyruvate and lactate in the urine can be due to sepsis-associated hypoperfusion and/or hypoxia [42]. In septic preterm infants, early metabolic responses include lactic acidosis and increases glucose requirements. A product of purine degradation called inosine was also observed in higher quantities in this condition because of cellular destruction. Trimethylamine N-oxide (TMAO) levels and certain vitamins such as riboflavin and nicotinamide were found to be reduced in neonatal septic patients [41].

*Clostridium difficile* is a spore-forming bacterial pathogen, which is the leading cause of healthcare-associated infective diarrhea [43, 44]. In the United States, more than 500,000 cases of *C. difficile* infection (CDI) are reported annually accruing \$6.3 billion in healthcare costs [44]. Intestinal dysbiosis is the reason for recurrent CDI. Urinary metabolites might be used as a prognostic method for patients with recurrent CDI as no diagnostic tests are available to predict the risk of CDI recurrences in patients [45]. With regards to *C. difficile* infections, Kao *et al.* performed NMR studies on urine of 31 infected subjects (age- and sex-matched to 31 healthy controls) and detected 53 metabolites. Choline appeared to be the most relevant for the diagnosis of *C. difficile* infection. This finding has been possibly attributed to the absence of choline-metabolizing microorganisms in this infection [33]. Isa *et al.* proposed four urinary metabolites as biomarkers for active tuberculosis patients [46]. Using an untargeted HPLC-MS and MS/MS, 102 urine samples were collected from infected individuals. The majority of the metabolites were neopterin, kynurenine, spermine, *N*-acetylated sugars, and sialic acids, which are host-derived metabolites involved in immune cell activation [46].

COVID 19 is a pneumonia caused by coronavirus (SARS-CoV 2) responsible for producing a global pandemic and still continues to be a worldwide emergency [47]. During SARS-CoV-2 infection, research studies have been conducted to correlate urinary constituent abnormalities for COVID-19 disease severity and outcome. A case study was done in four cohorts namely, healthy control (n = 27), non-COVID-19 control (n = 17), patients with nonsevere COVID-19 (n = 48), and patients with severe COVID-19 (n = 23) [48]. Peptide yields from urine samples in patients with severe and nonsevere cases were found to be greater compared to serum samples in the healthy control group. A total of 16,148 peptides and 1494 proteins were obtained from sera using tandem mass-tag (TMT)-based proteomics, while 19,732 peptides and 3854 proteins were identified from urine. Similarly, 80% of detectable serum proteins and 62% of serum metabolites were found in urine of the infected group. Cytoplasmic proteins (26%) and membrane proteins (21%) were the most abundant protein groups in the urinary proteome, whereas the proportion of secreted proteins was only 16%. Further, more intracellular compartment proteins released from tissues were seen in the urinary proteome of the infected group compared to the serum proteome. Also, 197 cytokines and their receptors were observed in urine of infected group, whereas 124 cytokines were seen in serum. In the same study, the reduction in endosomal sorting complexes required for transport (ESCRT) complex proteins and downregulation

of CXCL14 in urine was reported to be associated with an increase in SARS-CoV-2 replication [48]. In another study consisting of 142 infected volunteers and 104 healthy volunteers, urine samples were subjected to mass spectrometry. Significant alterations in nitrogen metabolism, D-glutamine and D-glutamate metabolism, aminoacyl-tRNA biosynthesis, arginine biosynthesis, glutathione metabolism, pantothenate and CoA biosynthesis, glyoxylate and dicarboxylate metabolism were noticed among infected group and control group. Nineteen amino acids such as alanine, leucine, glutamine, tryptophan, and 15 acylcarnitines were obtained from urine analysis. Glycine level was decreased in the infected group. Alteration in valine was also observed. This study suggested acylcarnitines as important markers for COVID-19 infection [49].

Acquired Immunodeficiency Syndrome (AIDS) is responsible for causing a severe immunosuppressive state on the immune system of humans. AIDS has emerged as a global health hazard and no effective methods are available for the characterization of affected patients. Urinary metabolomics can be a promising method to differentiate affected and nonaffected AIDS individuals, and also for monitoring the progress of HIV therapeutics. Studies using biofluids, such as urine, whole blood, and serum, have also been employed to identify metabolite markers correlating to HIV-induced oxidative stress (OS). Munshi et al. suggested that urinary neopterin could be used as a metabolic biomarker of AIDS infection. Urinary glutamic acid and formic acid levels were higher in HIV/AIDS patients compared to healthy controls. When comparing HIV-infected patients treated with or without antiretroviral therapy (ART), ART naïve patients had lower levels of urinary methionine, 2-methylglutaric acid, l-alanine, and glycolic acid, however, patients receiving ART had even reduced levels of the aforesaid metabolites [50]. Hence, urinary amino acids and their metabolites can help to serve as markers for assessing the progress of ART in AIDS patients.

Malaria is a mosquito-borne parasitic illness caused by *Plasmodium falciparum*, *Plasmodium vivax,* and *Plasmodium berghei*. Morbidity and mortality caused by these parasites are greater in tropical and sub-tropical countries. Prospect of infection biomarkers in biofluids is therefore important in a population to control the impact of the infection. Urinary metabolites could be used as a tool to differentiate the infected groups from the noninfected ones. In a case-control study of 21 *P. falciparum-*infected individuals and 25 controls, urine samples subjected to high-performance liquid chromatography-high resolution mass spectrometry (HPLC/HRMS) revealed altered levels of 1,3-diacetylpropane, *N*-acetylputrescine, and *N*-acetylspermidine between patients and control cohorts, thereby suggesting these molecules as potential biomarkers for malarial infections [51]. NMR spectroscopy of urinary samples from patients infected with *P. vivax* was also studied. Urinary ornithine and pipecolic acid were higher in malarial patients and could be used as a potential biomarker to differentiate between malarial and nonmalarial cases [52]. In another study using a mouse model of *P. berghei* infection, 4-amino-1-[3-hydroxy-5-(hydroxymethyl)-2,3-dihydrofuran-2-yl]pyrimidin-2(1H)-one and 2-amino-4-({[5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxy-4,5-dihydrofuran-2-yl]methyl}sulfanyl) butanoic acid were the two urinary metabolites detected in infected mice groups compared to healthy mice [53]. Therefore, the aforementioned catabolites in urine may aid to assess the progression of the disease among affected individuals.

#### **5.5 Renal dysfunction**

Acute kidney injury is defined as a sudden phase of kidney failure that occurs in a few hours. This condition is characterized by an increase in serum creatinine and a

#### *Application of Urine Metabolomics as a Marker in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.109808*

notable decrease in urine output [54]. Chronic kidney disease (CKD) is a condition, where glomerular filtration rate is progressively affected along with kidney damage [55]. In a study conducted in patients with acute kidney injury, urine samples from patients subjected to LC-MS and 1H NMR scanning revealed an increase in urea cycle, proline metabolism, nitric oxide pathway, and its metabolite, asymmetric dimethylarginine (ADMA), serotonin metabolism and homovanillic acid. On the other hand, a decrease in Kreb's cycle and citrate, benzoate metabolism and hippurate, pyruvate metabolism, and lactate were observed [56–58]. In another study conducted by Matin-Lorenzo et al., urine samples were collected from 24 control subjects and 38 patients with acute kidney injury. LC-MS/MS analysis revealed urinary 2-hydroxybutyric acid, pantothenic acid, and hippuric acid were significantly downregulated and urinary N-acetylneuraminic acid, phosphoethanolamine and serine were upregulated in diseased patients [59]. It was also reported that a low risk of chronic kidney disease is associated with urinary glycine and histidine, increased urinary lysine, and NG-monomethyl-L-arginine (NMMA) [60]. However, carnitine metabolism, betaoxidation and acylcarnitines, phenylacetylglutamine, urea cycle, proline metabolism and their metabolites, proline, and citrulline were increased in chronic kidney disease. Conversely, urea cycle, proline metabolism, nitric oxide pathway and ADMA, Krebs cycle and citrate, bile acid metabolism, and taurocholate were reported downregulated in chronic kidney disease conditions [61, 62].

Diabetes is a chronic (DM) metabolic disease that result in unusually higher preprandial plasma glucose levels as a result of defects in insulin secretion, insulin activity, or both. Chronic diabetes can lead to several pathophysiological conditions that range from cardiovascular abnormalities to renal failure. The hemodynamic dysregulation caused by diabetes mediates renal injury by inducing abnormal morphological and functional changes in the renal nephrons [63]. Gas chromatographymass spectrometry was used to quantify 94 urine metabolites in screening cohorts of patients with diabetes mellitus (DM) and chronic kidney disease CKD (DM + CKD), in patients with DM without CKD (DM–CKD), and in healthy controls. Thirteen metabolites were significantly reduced in the DM + CKD cohorts compared to healthy groups. Twelve of them are related to mitochondrial metabolism, suggesting a suppression of mitochondrial activity in diabetic kidney disease [64].

Autosomal dominant polycystic disease is a hereditary disorder which is characterized by cyst formation in ductal organs, mainly the kidney and the liver, and also by gastrointestinal, musculoskeletal, and cardiovascular abnormalities [65], Research conducted in a mouse model for autosomal dominant polycystic kidney disease was used to assess whether metabolomic shifts prior to renal cystogenesis can aid in early diagnosis for the condition. Utilizing GC-MS time of flight spectroscopy, urine samples collected from mice prior to exhibiting any serological evidence of kidney dysfunction revealed that purine and galactose metabolic pathways were affected, with elevation of biomarkers such as allantoic and adenosine [66].

#### **6. Conclusion**

Urine metabolomics is a powerful diagnostic technique that can be utilized to diagnose several diseases, including cancers, hereditary diseases, immune-mediated and metabolic disorders, and renal dysfunction. Urine metabolite constituents essentially serve as biomarker signatures to identify pathways related to specific diseases as well as to detect abnormal concentrations of the metabolites that may be associated with

the disease. Furthermore, the analysis of urinary metabolome can be used to evaluate disease activity, response to treatments and to monitor the progression or remission of the disease. Additionally, the standardization and curation of urine metabolomic databases for health and pathological phenotypes can potentially be developed and employed in routine clinical settings for disease diagnosis.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Abraham Joseph Pellissery1 , Poonam Gopika Vinayamohan2 , Leya Susan Viju3 , Divya Joseph3 and Kumar Venkitanarayanan3 \*

1 Department of Comparative, Diagnostic and Population Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA

2 Department of Preventive Medicine, College of Veterinary Medicine, Ohio State University, Colombus, OH, USA

3 Department of Animal Science, University of Connecticut. Storrs, CT, USA

\*Address all correspondence to: kumar.venkitanarayanan@uconn.edu

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Haber MH. Pisse prophecy: A brief history of urinalysis. Clinics in Laboratory Medicine. 1988;**8**:415-430

[2] Bolodeoku J, Donaldson D. Urinalysis in clinical diagnosis. Journal of Clinical Pathology. 1996;**49**:623-626

[3] Echeverry G, Hortin GL, Rai AJ. Introduction to urinalysis: Historical perspectives and clinical application. Methods in Molecular Biology. 2010;**641**:1-12. DOI: 10.1007/ 978-1-60761-711-2\_1

[4] Bouatra S, Aziat F, Mandal R, Guo AC, Wilson MR, Knox C, et al. The human urine metabolome. PLoS One. 2013;**8**. DOI: 10.1371/journal.pone.0073076

[5] Segers K, Declerck S, Mangelings D, Heyden YV, Eeckhaut AV. Analytical techniques for metabolomic studies: A review. Bioanalysis. 2019;**11**:2297-2318. DOI: 10.4155/bio-2019-0014

[6] Thévenot EA, Roux A, Xu Y, Ezan E, Junot C. Analysis of the human adult urinary metabolome variations with age, body mass index, and gender by implementing a comprehensive workflow for univariate and OPLS statistical analyses. Journal of Proteome Research. 2015;**14**:3322-3335. DOI: 10.1021/acs. jproteome.5b00354

[7] Ryan D, Robards K, Prenzler PD, Kendall M. Recent and potential developments in the analysis of urine: A review. Analytica Chimica Acta. 2011;**684**:17-29. DOI: 10.1016/j. aca.2010.10.035

[8] Zhang A, Sun H, Wu X, Wang X. Urine metabolomics. Clinica Chimica Acta. 2012;**414**:65-69. DOI: 10.1016/J. CCA.2012.08.016

[9] Chetwynd AJ, Dunn WB, Rodriguez-Blanco G. Collection and preparation of clinical samples for metabolomics. In: Advances in Experimental Medicine and Biology. Vol. 965. Springer New York LLC; 2017. pp. 19-44. DOI: 10.1007/978-3-319- 47656-8\_2

[10] Idle JR, Gonzalez FJ. Metabolomics. Cell Metabolism. 2007;**6**:348-351. DOI: 10.1016/j.cmet.2007.10.005

[11] Lichtenberg S, Trifonova OP, Maslov DL, Balashova EE, Lokhov PG. Metabolomic laboratory-developed tests: Current status and perspectives. Metabolites. 2021;**11**:423. DOI: 10.3390/ metabo11070423

[12] Ren S, Hinzman AA, Kang EL, Szczesniak RD, Lu LJ. Computational and statistical analysis of metabolomics data. Metabolomics. 2015;**11**:1492-1513. DOI: 10.1007/s11306-015-0823-6

[13] González-Domínguez R, González-Domínguez Á, Sayago A, Fernández-Recamales Á. Recommendations and best practices for standardizing the pre-analytical processing of blood and urine samples in metabolomics. Metabolites. 2020;**10**:1-18. DOI: 10.3390/metabo10060229

[14] Roberts LD, Gerszten RE. Toward new biomarkers of cardiometabolic diseases. Cell Metabolism. 2013;**18**:43-50. DOI: 10.1016/j.cmet.2013.05.009

[15] Kind T, Tolstikov V, Fiehn O, Weiss RH. A comprehensive urinary metabolomic approach for identifying kidney cancer. Analytical Biochemistry. 2007;**363**:185-195. DOI: 10.1016/j. ab.2007.01.028

[16] Kim K, Aronov P, Zakharkin SO, Anderson D, Perroud B, Thompson IM, et al. Urine metabolomics analysis for kidney cancer detection and biomarker discovery. Molecular and Cellular Proteomics. 2009;**8**:558-570. DOI: 10.1074/mcp.M800165-MCP200

[17] Wang X, Zhang A, Sun H. Power of metabolomics in diagnosis and biomarker discovery of hepatocellular carcinoma. Hepatology. 2013;**57**:2072- 2077. DOI: 10.1002/hep.26130

[18] Cornier MA, Dabelea D, Hernandez TL, Lindstrom RC, Steig AJ, Stob NR, et al. The metabolic syndrome. Endocrine Reviews. 2008;**29**:777-822. DOI: 10.1210/er.2008-0024

[19] Wu Q, Li J, Sun X, He D, Cheng Z, Li J, et al. Multi-stage metabolomics and genetic analyses identified metabolite biomarkers of metabolic syndrome and their genetic determinants. eBioMedicine. 2021;**74**. DOI: 10.1016/j. ebiom.2021.103707

[20] Wang X, Zhang A, Han Y, Wang P, Sun H, Song G, et al. Urine metabolomics analysis for biomarker discovery and detection of jaundice syndrome in patients with liver disease. Molecular and Cellular Proteomics. 2012;**11**:370-380. DOI: 10.1074/mcp.M111.016006

[21] Dong S, Zhan ZY, Cao HY, Wu C, Bian YQ, Li JY, et al. Urinary metabolomics analysis identifies key biomarkers of different stages of nonalcoholic fatty liver disease. World Journal of Gastroenterology. 2017;**23**:2771-2784. DOI: 10.3748/wjg.v23. i15.2771

[22] Men L, Pi Z, Zhou Y, Wei M, Liu Y, Song F, et al. Urine metabolomics of high-fat diet induced obesity using UHPLC-Q-TOF-MS. Journal of Pharmaceutical and Biomedical Analysis. 2017;**132**:258-266. DOI: 10.1016/j. jpba.2016.10.012

[23] Alonso A, Julià A, Vinaixa M, Domènech E, Fernández-Nebro A, Cañete JD, et al. Urine metabolome profiling of immune-mediated inflammatory diseases. BMC Medicine. 2016;**14**:133. DOI: 10.1186/ s12916-016-0681-8

[24] Stephens NS, Siffledeen J, Su X, Murdoch TB, Fedorak RN, Slupsky CM. Urinary NMR metabolomic profiles discriminate inflammatory bowel disease from healthy. Journal of Crohn's and Colitis. 2013;**7**. DOI: 10.1016/j. crohns.2012.04.019

[25] Dawiskiba T, Deja S, Mulak A, Zabek A, Jawień E, Pawełka D, et al. Serum and urine metabolomic fingerprinting in diagnostics of inflammatory bowel diseases. World Journal of Gastroenterology. 2014;**20**:163-174. DOI: 10.3748/wjg.v20. i1.163

[26] Williams HR, Cox JI, Walker DG, North BV, Patel VM, Marshall SE, et al. Characterization of inflammatory bowel disease with urinary metabolic profiling. Official Journal of the American College of Gastroenterology|ACG. 2009;**104**:1435-1444

[27] Keshteli AH, Tso R, Dieleman LA, Park H, Kroeker KI, Jovel J, et al. A distinctive urinary metabolomic fingerprint is linked with endoscopic postoperative disease recurrence in Crohn's disease patients. Inflammatory Bowel Diseases. 2018;**24**:861-870. DOI: 10.1093/ibd/izx070

[28] Yamamoto M, Shanmuganathan M, Hart L, Pai N, Britz-McKibbin P. Urinary metabolites enable differential diagnosis and therapeutic monitoring of Pediatric inflammatory bowel disease.

*Application of Urine Metabolomics as a Marker in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.109808*

Metabolites. 2021;**11**:245. DOI: 10.3390/ metabo11040245

[29] Reilly IA, Doran JB, Smith B, FitzGerald GA. Increased thromboxane biosynthesis in a human preparation of platelet activation: Biochemical and functional consequences of selective inhibition of thromboxane synthase. Circulation. 1986;**73**:1300-1309. DOI: 10.1161/01.cir.73.6.1300

[30] Li X, Yang S, Qiu Y, Zhao T, Chen T, Su M, et al. Urinary metabolomics as a potentially novel diagnostic and stratification tool for knee osteoarthritis. Metabolomics. 2010;**6**:109-118. DOI: 10.1007/s11306-009-0184-0

[31] Vignoli A, Rodio DM, Bellizzi A, Sobolev AP, Anzivino E, Mischitelli M, et al. NMR-based metabolomic approach to study urine samples of chronic inflammatory rheumatic disease patients. Analytical and Bioanalytical Chemistry. 2017;**409**:1405-1413. DOI: 10.1007/ s00216-016-0074-z

[32] Zou W, Wen X, Sheng X, Zheng Y, Xiao Z, Luo J, et al. Gas chromatographymass spectrometric method-based urine metabolomic profile of rats with pelvic inflammatory disease. Experimental and Therapeutic Medicine. 2016;**11**:1653- 1660. DOI: 10.3892/etm.2016.3142

[33] Tounta V, Liu Y, Cheyne A, Larrouy-Maumus G. Metabolomics in infectious diseases and drug discovery. Molecular Omics. 2021;**17**:376-393. DOI: 10.1039/d1mo00017a

[34] Gupta A, Bansal N, Houston B. Metabolomics of urinary tract infection: A new uroscope in town. Expert Review of Molecular Diagnostics. 2012;**12**:361- 370. DOI: 10.1586/erm.12.27

[35] Lv H, Hung CS, Chaturvedi KS, Hooton TM, Henderson JP. Development of an integrated metabolomic profiling approach for infectious diseases research. Analyst. 2011;**136**:4752-4763. DOI: 10.1039/c1an15590c

[36] Pacchiarotta T, Hensbergen PJ, Wuhrer M, Van Nieuwkoop C, Nevedomskaya E, Derks RJE, et al. Fibrinogen alpha chain O-glycopeptides as possible markers of urinary tract infection. Journal of Proteomics. 2012;**75**:1067-1073. DOI: 10.1016/j. jprot.2011.10.021

[37] Gupta A, Dwivedi M, Gowda GAN, Mahdi AA, Jain A, Ayyagari A, et al. 1H NMR spectroscopy in the diagnosis of Klebsiella pneumoniae-induced urinary tract infection. NMR in Biomedicine. 2006;**19**:1055-1061. DOI: 10.1002/ nbm.1078

[38] Pacchiarotta T, Deelder AM, Mayboroda OA. Metabolomic investigations of human infections. Bioanalysis. 2012;**4**:919-925. DOI: 10.4155/bio.12.61

[39] Gupta A, Dwivedi M, Mahdi AA, Gowda GAN, Khetrapal CL, Bhandari M. 1H-nuclear magnetic resonance spectroscopy for identifying and quantifying common uropathogens: A metabolic approach to the urinary tract infection. BJU International. 2009;**104**:236-244. DOI: 10.1111/j. 1464-410X.2009.08448.x

[40] Slupsky CM, Rankin KN, Fu H, Chang D, Rowe BH, Charles PGP, et al. Pneumococcal pneumonia: Potential for diagnosis through a urinary metabolic profile. Journal of Proteome Research. 2009;**8**:5550-5558. DOI: 10.1021/ pr9006427

[41] Sarafidis K, Chatziioannou AC, Thomaidou A, Gika H, Mikros E, Benaki D, et al. Urine metabolomics in neonates with late-onset sepsis in a case-control study. Scientific Reports. 2017;**7**. DOI: 10.1038/srep45506

[42] Su L, Li H, Xie A, Liu D, Rao W, Lan L, et al. Dynamic changes in amino acid concentration profiles in patients with sepsis. PLoS One. 2015;**10**. DOI: 10.1371/journal.pone.0121933

[43] Smits WK, Lyras D, Lacy DB, Wilcox MH, Kuijper EJ. Clostridium difficile infection. Nature Reviews Disease Primers. 2016;**2**:1-20. DOI: 10.1038/nrdp.2016.20

[44] Pellissery AJ, Vinayamohan PG, Yin HB, Mooyottu S, Venkitanarayanan K. In vitro efficacy of sodium selenite in reducing toxin production, spore outgrowth and antibiotic resistance in hypervirulent clostridium difficile. Journal of Medical Microbiology. 2019;**68**:1118-1128. DOI: 10.1099/jmm.0.001008

[45] Kao D, Ismond KP, Tso V, Millan B, Hotte N, Fedorak RN. Urinebased metabolomic analysis of patients with Clostridium difficile infection: A pilot study. Metabolomics. 2016;**12**. DOI: 10.1007/s11306-016-1080-z

[46] Isa F, Collins S, Lee MH, Decome D, Dorvil N, Joseph P, et al. Mass spectrometric identification of urinary biomarkers of pulmonary tuberculosis. eBioMedicine. 2018;**31**:157-165. DOI: 10.1016/j.ebiom.2018.04.014

[47] Yang L, Liu S, Liu J, Zhang Z, Wan X, Huang B, et al. COVID-19: Immunopathogenesis and Immunotherapeutics. Signal transduction and targeted. Therapy. 2020;**5**. DOI: 10.1038/ s41392-020-00243-2

[48] Bi X, Liu W, Ding X, Liang S, Zheng Y, Zhu X, et al. Proteomic and metabolomic profiling of urine uncovers immune responses in patients with COVID-19. Cell Reports. 2022;**38**. DOI: 10.1016/j.celrep.2021.110271

[49] Barberis E, Timo S, Amede E, Vanella VV, Puricelli C, Cappellano G, et al. Large-scale plasma analysis revealed new mechanisms and molecules associated with the host response to SARS-CoV-2. International Journal of Molecular Sciences. 2020;**21**:8623. DOI: 10.3390/ijms21228623

[50] Munshi SU, Rewari BB, Bhavesh NS, Jameel S. Nuclear magnetic resonance based profiling of biofluids reveals metabolic dysregulation in HIV-infected persons and those on anti-retroviral therapy. PLoS One. 2013;**8**. DOI: 10.1371/ journal.pone.0064298

[51] Abdelrazig S, Ortori CA, Davey G, Deressa W, Mulleta D, Barrett DA, et al. A metabolomic analytical approach permits identification of urinary biomarkers for *Plasmodium falciparum* infection: A case-control study. Malaria Journal. 2017;**16**:1-8. DOI: 10.1186/ s12936-017-1875-z

[52] Sengupta A, Ghosh S, Basant A, Malusare S, Johri P, Pathak S, et al. Global host metabolic response to *Plasmodium vivax* infection: A 1H NMR based urinary metabonomic study. Malaria Journal. 2011;**10**. DOI: 10.1186/1475-2875-10-384

[53] Tritten L, Keiser J, Godejohann M, Utzinger J, Vargas M, Beckonert O, et al. Metabolic profiling framework for discovery of candidate diagnostic markers of malaria. Scientific Reports. 2013;**3**. DOI: 10.1038/srep02769

[54] Chawla LS, Eggers PW, Star RA, Kimmel PL. Acute kidney injury and chronic kidney disease as interconnected syndromes. New England Journal of Medicine. 2014;**371**:58-66. DOI: 10.1056/ nejmra1214243

*Application of Urine Metabolomics as a Marker in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.109808*

[55] Webster AC, Nagler EV, Morton RL, Masson P. Chronic kidney disease. Lancet. 2017;**389**:1238-1252. DOI: 10.1016/S0140-6736(16)32064-5

[56] Barrios C, Spector TD, Menni C. Blood, urine and faecal metabolite profiles in the study of adult renal disease. Archives of Biochemistry and Biophysics. 2016;**589**:81-92. DOI: 10.1016/j.abb.2015.10.006

[57] Klawitter J, Haschke M, Kahle C, Dingmann C, Klawitter J, Leibfritz D, et al. Toxicodynamic effects of ciclosporin are reflected by metabolite profiles in the urine of healthy individuals after a single dose. British Journal of Clinical Pharmacology. 2010;**70**:241-251. DOI: 10.1111/j. 1365-2125.2010.03689.x

[58] Bairaktari E, Seferiadis K, Liamis G, Psihogios N, Tsolas O, Elisaf M. Rhabdomyolysis-related renal tubular damage studied by proton nuclear magnetic resonance spectroscopy of urine. Clinical Chemistry. 2002;**48**:1106-1109

[59] Martin-Lorenzo M, Gonzalez-Calero L, Ramos-Barron A, Sanchez-Niño MD, Gomez-Alamillo C, García-Segura JM, et al. Urine metabolomics insight into acute kidney injury point to oxidative stress disruptions in energy generation and H2S availability. Journal of Molecular Medicine. 2017;**95**:1399-1409. DOI: 10.1007/s00109-017-1594-5

[60] McMahon GM, Hwang SJ, Clish CB, Tin A, Yang Q, Larson MG, et al. Urinary metabolites along with common and rare genetic variations are associated with incident chronic kidney disease. Kidney International. 2017;**91**:1426-1435. DOI: 10.1016/j.kint.2017.01.007

[61] Duranton F, Lundin U, Gayrard N, Mischak H, Aparicio M, Mourad G,

et al. Plasma and urinary amino acid metabolomic profiling in patients with different levels of kidney function. Clinical Journal of the American Society of Nephrology. 2014;**9**:37-45. DOI: 10.2215/CJN.06000613

[62] Nkuipou-Kenfack E, Duranton F, Gayrard N, Argilés À, Lundin U, Weinberger KM, et al. Assessment of metabolomic and proteomic biomarkers in detection and prognosis of progression of renal function in chronic kidney disease. PLoS One. 2014;**9**. DOI: 10.1371/ journal.pone.0096955

[63] Fakhruddin S, Alanazi W, Jackson KE. Diabetes-induced reactive oxygen species: Mechanism of their generation and role in renal injury. Journal Diabetes Research. 2017;**2017**:8379327. DOI: 10.1155/ 2017/8379327

[64] Sharma K, Karl B, Mathew AV, Gangoiti JA, Wassel CL, Saito R, et al. Metabolomics reveals signature of mitochondrial dysfunction in diabetic kidney disease. Journal of the American Society of Nephrology. 2013;**24**:1901- 1912. DOI: 10.1681/ASN.2013020126

[65] Gabow PA. Polycystic kidney disease: Clues to pathogenesis. Kidney International. 1991;**40**:989-996. DOI: 10.1038/ki.1991.306

[66] Taylor SL, Ganti S, Bukanov NO, Chapman A, Fiehn O, Osier M, et al. A metabolomics approach using juvenile cystic mice to identify urinary biomarkers and altered pathways in polycystic kidney disease. American Journal of Physiology. Renal Physiology. 2010;**298**:909-922. DOI: 10.1152/ ajprenal.00722.2009.-Autosomal

### **Chapter 6**

## Detecting Naloxone in Adulterated Urine Samples: Can Naloxone Be Detected When Buprenorphine/ Naloxone Film Is Dipped Directly into Urine and Water?

*Hiroko Furo, Tony Lin, Yi Yuan Zhou and Sarah Abdelsayed*

#### **Abstract**

This study is aimed at exploring if "naloxone" is detected in urine and water samples by dipping buprenorphine/naloxone film directly into these specimens. This study utilized 12 urine samples from 12 healthy participants who were not taking any medications with four samples added as a control. Sublingual generic buprenorphine/naloxone (8 mg/2 mg) film was dipped directly into these samples. They were sent to the ARUP laboratory for gas chromatography-mass spectrometry (GC/MS) quantitative analysis. The results were analyzed using IBM SPSS Statistics software. The results showed that "naloxone" was detected at high levels both in urine samples and in water, into which buprenorphine/naloxone film was dipped. In addition, the "naloxone" level was associated with the area of the film and the time in contact with the urine or water samples, but it was not affected by the urine concentration or the temperature of the specimens. This information will be useful for clinicians in identifying urine manipulation and interpreting urine drug test results and can help them for accurate monitoring of their patients' treatment progress in opioid use disorder (OUD) treatment programs.

**Keywords:** opioid, urine, buprenorphine, norbuprenorphine, creatinine

#### **1. Introduction**

Naloxone\* is an opioid antagonist that binds to *mu*-opioid receptors and blocks opioid agonist effect. It is primarily metabolized by the liver and excreted by the kidneys. In the liver, it predominantly undergoes glucuronide conjugation to "naloxone-β-3-glucuronide", while minor metabolic pathways generate nornaloxone

<sup>\*</sup> In order to differentiate "naloxone" found in urine versus other naloxone format such as naloxone in buprenorphine/naloxone combined medication, "naloxone" and other components found in specimens are expressed with quotation marks around them as "naloxone."

(noroxymorphone) through N-dealkylation of naloxone by CYP2C18 and 2C19 primarily, which go through subsequent glucuronidation [1, 2].

Naloxone has approximately 30–45 minute duration of action with a short halflife of 1.87–5.45 hours and reaches peak activity within 0.750–1.13 hours when taken sublingually [2–4]. It dissociates rapidly from opioid receptors within 6.5 minutes and has a low bioavailability of an estimated 3% through the sublingual route of administration with extensive first-pass metabolism [5]. Naloxone can be administered through various routes, which include intravenous, intramuscular, subcutaneous, endotracheal, sublingual, intralingual, submental and intranasal routes [6]. Naloxone, however, is estimated to be 50–250 times more potent when intravenously injected than orally administered [7]. Given this unique characteristic, naloxone is administered parenterally in order to increase its bioavailability, especially when it is used to reverse opioid effect in opioid overdose cases [8].

The effect of naloxone in combined buprenorphine/naloxone medication when taken sublingually is assumed to be clinically insignificant due to its poor absorption (<10%) and extensive first pass metabolism [4]. Therefore, combining buprenorphine and naloxone is strategized to deter unintended use such as intravenous injection and intranasal insufflation [9]. When taken through these routes, the addition of naloxone to buprenorphine antagonizes *mu*-opioid receptors and prevent the intoxicating effects of buprenorphine; however, there are some controversies in adding naloxone to buprenorphine products [5, 10].

While it is widely accepted that naloxone has a poor oral and sublingual bioavailability, many studies found "naloxone" detected at various levels in urine samples of patients who are on sublingual buprenorphine/naloxone medication [11, 12]. For example, Strickland and Burson found that 92.7% of urine samples from those who were on buprenorphine/naloxone medication had >30 ng/mL of "naloxone" detected [13]. At high levels within the system, naloxone may exert its antagonist effect on *mu*-opioid receptors, competing with the desired therapeutic effect of buprenorphine [7]. Therefore, it has been argued that combined medications can still contribute to certain negative effects of naloxone such as precipitated withdrawal during buprenorphine/naloxone induction or maintenance phase of OUD treatment [14].

In addition, many means of urine adulteration among the OUD patients who are on buprenorphine have been reported. One of the common methods of urine adulteration is "dipping" or "spiking" in which patients attempt to dissolve buprenorphine/naloxone film into urine samples directly. Heikman et al. reported that stable patients who are on buprenorphine/naloxone treatment had a median urine "naloxone" concentration of 60 ng/mL while that of unstable patients had a similar median of 70 ng/mL. The same study also found that the urine "naloxone" concentration in unstable patients ranged between 10 and 1700 ng/mL while that of the stable patients had a narrower window between 5 and 200 ng/mL [15]. This contrasting comparison of urine "naloxone" concentrations between stable and unstable patients raises the question of appropriate use of buprenorphine/naloxone therapy and the possibility of high "naloxone" levels in urine as an indication for urine adulteration.

Urine testing strategies have been developed to address this practice. Many previous studies discussed the use of elevated ratio between "buprenorphine" and "norbuprenorphine" in the urine as an indicator of urine adulteration [16–19]. In addition to this high ratio between "buprenorphine" to "norbuprenorphine", high "naloxone" levels in urine samples may also point to adulteration. Warrington, et al. suggested that urine samples with "naloxone" concentration greater than 2000 ng/mL should

*Detecting Naloxone in Adulterated Urine Samples: Can Naloxone Be Detected When… DOI: http://dx.doi.org/10.5772/intechopen.109412*

raise suspicion of adulteration, likely from dipping buprenorphine/naloxone film into the sample [20]. In their study, "buprenorphine" to "norbuprenorphine" ratios were found to be high along with the elevated "naloxone" levels in the urine samples with suspected adulteration. This supports the notion that "naloxone" concentration in the urine can be used as one of the indicators for urine sample adulteration.

While previous studies utilized urine samples from individuals who were on prescribed buprenorphine/naloxone, the impact of adulteration on substance-free urine and purified water remains unproven. Burns et al. conducted an in vitro study in which naloxone was added to urine samples that came from healthy volunteers who were not on any medications, and they found that "naloxone" was detected in the urine samples [21]. While the elevated "naloxone" level in urine samples may be flagged for adulteration, to our knowledge, there are no studies to date that demonstrate whether high levels of "naloxone" can be detected by dipping buprenorphine/ naloxone film in water or urine samples from those who are not taking the combination medication.

The aim of this study is to detect and quantify the presence of "naloxone" in adulterated urine samples from those who are not on buprenorphine/naloxone medication and adulterated purified water samples. The findings of this study should deepen our understanding on the pharmacokinetics and pharmacodynamics of naloxone.

#### **2. Methods**

#### **2.1 Data**

This is a urine test experiment study. After the Institutional Review Board (IRB) application was approved at the University of Texas Health at San Antonio (IRB Protocol ID 20220593HU), 12 urine samples from 12 participants were collected at our clinic. These participants were recruited through flyers and word-of-mouth. One of the inclusion criteria stipulates that the participant is not taking any medications or illegal substances such as opioids and marijuana. This criterion was included because certain medications and substances can inhibit or induce relevant CYP450, which might affect naloxone metabolism and consequently the "naloxone" levels in urine [12]. Another inclusion criterion was "healthy" participants without any major health issues. This inclusion criterion was added because naloxone is metabolized in the liver through glucuronide conjugation, and hepatic impairment can alter naloxone metabolism and consequently affect "naloxone" levels in the urine [22]. The last inclusion criterion was the ability to provide >80 mL urine samples. 15 healthy participants who were not on any medications were initially recruited. 3 participants from this initial pool were excluded due to their inability to provide adequate urine volume. Therefore, 12 urine samples from 12 participants were included in this study. At the time of urine collection, these 12 participants were asked to fill out a demographic information form. Their participation to this study was compensated financially for their time and urine sample contribution.

After the 12-urine samples were collected, each sample was then divided into four specimen aliquots with 20 mL each. Then, sublingual generic buprenorphine/ naloxone (8 mg/2 mg) film of 12.8 mm width and 22.0 mm length was dipped directly into each urine specimen. In the first aliquot, 1 mm of buprenorphine/naloxone film was vertically dipped into the urine specimen for three seconds (1 mm\*3 sec); in the second, half of a film, approximately 11 mm, was vertically dipped for three seconds

(half\*3 sec); in the third, a full film was dipped for three seconds (full\*3 sec); and in the fourth, a full film was dipped for thirty seconds (full\*30 sec). That way, we could investigate if area and/or duration of dipping can alter the "naloxone" levels. In addition, four control samples were utilized; (1) room temperature purified water (RT water), (2) water at approximately 97o F (Body Temperature or BT water), (3) 2 mL of urine diluted with 18 mL RT water (10% RT), and (4) 2 mL of urine diluted with BT 18 mL water (10% BT). These four control samples were added to examine if "naloxone" can be detected in water without any human urine and if the temperature and concentration of urine can affect "naloxone" levels in adulterated urine samples.

#### **2.2 Data analysis**

All specimens mentioned above were sent to the ARUP laboratory for quantitative tests. The tests included "naloxone" and "creatinine" levels. The results were stored in **Microsoft Excel (Microsoft 365)** without any identification of the participants. Then, the data sets were analyzed with T tests/Analysis of Variance (ANOVA) depending on the data sets, utilizing IBM SPSS statistics software (Version 28.0.0.0) [23]. The "alpha level" was set as 0.05 for the analyses (α = 0.05).

#### **3. Results**

The demographic information of the 12 participants was reviewed and is summarized in **Table 1**.

The majority of the participants were male (83.3%), Hispanic (66.7%), nonsmoker (91.7%), and non-veterans (91.7%) with some college education (66.7%). All of them denied any medication use or current medical issues. The average age of the 12 participants was 32.9 years old with a range of 20 to 57 years old. The average BMI was 29.1 kg/m2 with a range of 21 to 40.3 kg/m2 .

The "naloxone" and "creatinine" levels were checked, and the average and standard deviation (SD) of the four different specimen aliquots in each sample are listed in **Table 2**. The "naloxone" level with maximum measurable level was 1000 ng/mL, and any values surpassing it were considered as 1000 ng/mL for the purpose of calculations. Similarly, when the "naloxone" level was lower than the minimal measurable or detectable level (<100 ng/mL), 0 ng/mL was used for the purpose of calculations. The reportable range of "creatinine" was 5-2239 mg/dL, while that of "naloxone" was 100-1000 ng/mL. The "naloxone" was reported with the unit of ng/mL while that of "creatinine" with mg/dL.

**Table 2** shows that when buprenorphine/naloxone film was dipped directly into these specimens, "naloxone" was detected in the samples. Although "naloxone" was not detected in one of the 1 mm\*3 sec specimens, all of the other specimens had "naloxone" detected in the urine specimens. In particular, all of the specimens with full film dipped for 30 seconds showed high levels of "naloxone" detected with >1000 ng/ mL. All the creatinine levels were within normal range (20–400 mg/dL in the ARUP laboratory report) with an average of 89.75 mg/dL.

A statistically significant difference is present when we compared with 1 mm dipped for 3 seconds, half film dipped for 3 seconds, and full film dipped for 3 seconds by a one-way ANOVA (F(2, 33) = [3.401], p = .045). This indicates that the larger the area of film in contact with the urine specimen, the higher the level of "naloxone" detected in the urine. Also, when we compare the specimens that had a full film dipped for 3 seconds (M = 765.83, SD = 356.91) versus 30 seconds (M = 1000, *Detecting Naloxone in Adulterated Urine Samples: Can Naloxone Be Detected When… DOI: http://dx.doi.org/10.5772/intechopen.109412*


*The information is presented as mean ± standard deviation (SD) or mean with n (number) in %. BMI, body mass index.*

#### **Table 1.**

*Demographic information of 12 participants.*


*Aliquot 1 (1 mm\*3 sec) = a buprenorphine/naloxone film was dipped vertically 1 mm for three seconds; Aliquot 2 (half\*3 sec) = half film was dipped for three seconds; Aliquot 3 (full\*3 sec) = full film was dipped for three seconds; and Aliquot 4 (full\*30 sec) = full film was dipped for 30 seconds. SD = Standard Deviation.*

#### **Table 2.**

*"Naloxone" levels in 12 urine samples.*

SD = 0) by an independent sample T test, a statistically significant difference of "naloxone" levels was observed; t(22) = −2.273, p = .033). This indicates the longer the time the film was dipped, the higher the "naloxone" levels were detected. Thus, these results indicate that the volume and duration of dipping can influence "naloxone" levels in urine.

Next, the average of "naloxone" levels in the 12 urine specimens, the diluted 10% urine sample, and purified water sample were compared. The results are summarized in **Table 3**. In the table below, "naloxone", "creatinine" and "naloxone/creatinine" levels are listed with the units and the cut off levels. Because the unit of "naloxone" was recorded with "ng/mL" while that of "creatinine" was (mg/dL), the ratio between "naloxone" and "creatinine" was listed with (\*−4) for an easier understanding.

**Table 3** shows that when the film was dipped into purified water without human urine, "naloxone" was detected. This indicates that "naloxone" detected in urine might be mere dissolvents instead of metabolites. Next, the "naloxone" levels of room


*ND (not detected) indicates that the "naloxone" or "creatinine" was under the detectable level. NA (not applicable) indicates that "naloxone" or "creatinine" was undetected, and thus we were not able to calculate "naloxone"/"creatinine" ratio. BT (body temperature) means that the sample temperature was ~97o F. RT (room temperature) in 10% RT means that the samples were diluted by room temperature purified water. The unit as well as the minimal and maximal measurable levels are listed in the table. "Creatinine" levels of four specimens in one sample are the same because only one urine "creatinine" level in each sample was tested.*

#### **Table 3.**

*"Naloxone" levels in urine specimens, 10% diluted urine, and purified water.*

*Detecting Naloxone in Adulterated Urine Samples: Can Naloxone Be Detected When… DOI: http://dx.doi.org/10.5772/intechopen.109412*

temperature water were compared with those of body temperature with one-sample T-test. We found that the "naloxone" levels of room temperature water (M = 836.75, SD = 326.50) were not significantly different from those with body temperature water (M = 909.25, SD = 181.50); t(6) = .388, p = .711). Thus, the temperature difference did not affect "naloxone" levels. Finally, when the "naloxone" levels of body temperature purified water, body temperature 10% urine and body temperature pure urine were compared by a one-way ANOVA, there was no statistical difference (F(2, 9) = [.690], p = .526). Thus, the urine concentration difference did not affect "naloxone" levels. These results indicate that temperature and concentration of urine samples did not affects "naloxone" levels.

#### **4. Discussion**

#### **4.1 Clinical effects of sublingual naloxone**

This study demonstrated that high levels of "naloxone" were found in adulterated urine samples from individuals who were not taking any medications. High levels of "naloxone" were also detected in the purified water samples when buprenorphine/ naloxone film was dipped into the samples. Thus, these results and those in the previous studies on urine "naloxone" levels suggest that "naloxone" can be detected in adulterated urine samples both from those who are not on buprenorphine/naloxone and those who are on buprenorphine/naloxone [15, 20]. This information can lead to the speculation that "naloxone" detected in urine samples can be mere dissolvents of some fraction of naloxone instead of metabolites, and consequently imply a possibility that "naloxone" in urine samples from the patients taking buprenorphine/ naloxone films were the mere dissolvents of some naloxone in the urinary system, considering naloxone's low bioavailability and extensive first-pass metabolism. Unfortunately, we were unable to distinguish between "naloxone" in urine that has been excreted by nephrons after it was absorbed and metabolized versus some fractions of "naloxone" that have been only dissolved in the system.

The clinical effects of sublingual naloxone on healthy patients have been controversial. For example, Nasser, et al. conducted a research study, in which 43 participants received one single sublingual tablet of brand name Suboxone (buprenorphine/ naloxone 2 mg/0.5 mg) and their plasma "naloxone" levels in their blood samples were monitored for up to 168 hours. They found that the participants with severe hepatic impairment had higher and longer "naloxone" levels detected compared with the healthy participants; however, the inactive metabolite, "naloxone 3-β-Dglucuronide" levels were similar between the two groups. They concluded that sublingual buprenorphine/naloxone combined product should be avoided for those who have severe hepatic impairment [4]. This study also suggested that naloxone is metabolized in the liver and may provide clinical effectiveness only for those who have hepatic impairment. The clinical effects of sublingual naloxone on healthy patients remain controversial.

Strickland and Burson reviewed the charts of 561 patients, 11.1% of whom were on buprenorphine/naloxone combination products while the others received mono products. The authors reported that urine "naloxone" was detected in the majority of samples; 97.8% from the 63 patients who were on the dual medication had >1 ng/ mL naloxone, and 92.7% of them had 30 ng/mL of naloxone. The authors argued that sublingual naloxone may be absorbed and thus cause unpleasant adverse effects [13].

By contrast, the pharmacological effects of sublingual naloxone have a 10-times lower binding affinity to μ opioid receptors, compared to buprenorphine, namely, rapid opioid receptor dissociation (approximately 6.5 minutes); short half-life of naloxone (60–90 minutes compared to 24–60 hours of buprenorphine); low bioavailability (estimated 3% sublingually); and first-pass bioavailablility, led to the postulation of insignificant clinical effects in sublingual naloxone. After the Food and Drug Administration (FDA) approved buprenorphine/naloxone combination medication in 2002 [24], this combined medication became the standard of care, especially after the Substance Abuse and Mental Health Services Administration (SAMHSA) warned about the misuse potentials of monotherapy [25]. However, the controversy of combined naloxone/buprenorphine versus monotherapy of buprenorphine has yet to be resolved, and further research and discussion on this topic await.

#### **4.2 Adulteration**

This study found that the amount of the film that was used to adulterated urine specimen did affect the concentration of "naloxone" detected in the urine. Submerging a small portion of the film for a short period of time (1 mm\*3 sec) in this study resulted in an average of 492 ng/mL of "naloxone" levels in the urine samples among the 12 participants. By contrast, the urine samples with full film dipped into the aliquot for 3 seconds (Full\*3 sec) yielded an average of 765.83 ng/mL of "naloxone". These numbers are very similar with those found in the study done by Warrington, et al. on the "naloxone" levels in suspected adulterated urine. They retrospectively reviewed "naloxone" levels of 1223 urine samples from two practice sites and reported that the average "naloxone" level was "633.65ng/mL (range 1-12,161 ng/mL) with 54% of samples <300 ng/mL and 8.0% having >2000ng/ mL. One of the sites had increased evidence of urine adulteration and 9.3% of the samples from this site contained > 2000 ng/ml of "naloxone" with an average of 686.8 ng/mL. The other site had no report of urine adulteration and demonstrated an average "naloxone" level of 570.9 ng/mL with 6.4% of samples containing > 2000 ng/mL. This study concluded that extremely high levels of "naloxone" can suggest urine adulteration [20]. Furo also reviewed 97 patient charts with urine drug screening results in an outpatient telemedicine OUD clinic and found that average "naloxone" level was 687.2 ng/mL that ranged from 5 ng/mL to>2000 ng/ mL with 15.30% >2000 ng/mL [26].

Heikman, et al. collected 40 urine samples from 32 patients and found much lower average of "naloxone" levels in their study. In their study design, the first group (Group 0: pre-treatment) received parental buprenorphine before buprenorphine/ naloxone treatment; the second group (Group 1: stable patients) consisted of stable patients who were on prescribed sublingual buprenorphine/naloxone treatment without any illicit substances in their urine; and the third group (Group 2: unstable patients) had prescribed sublingual buprenorphine/naloxone with unexpected urine test results. The urine samples were collected about 24 hours after the last buprenorphine/naloxone medication dispensation. The study found that the median naloxone level in the stable phase was 60 μg/L (=ng/mL, henceforth ng/mL), ranged from 5 ng/ mL to 200 ng/mL while that in the unstable phase was 70 ng/mL, ranged from 70 ng/ mL to 1700 ng/mL. They concluded that high "naloxone" level can indicate noncompliance of buprenorphine/naloxone treatment [15].

The average "naloxone" levels in the study by Heikman, et al., [15] was much lower compared with those in the studies mentioned above. This may be because *Detecting Naloxone in Adulterated Urine Samples: Can Naloxone Be Detected When… DOI: http://dx.doi.org/10.5772/intechopen.109412*

naloxone has a short half-life of 60–90 minutes [22] and because their urine collection was done approximately 24 hours after the last dose of buprenorphine/naloxone medication. The average "naloxone" levels of this current study (e.g. 492 ng/mL of 1 mm\*3 sec specimen group and 765.83 ng/mL of full\*3 sec group) of the adulterated urine samples from those who were not on buprenorphine/naloxone were similar to those of these previous studies (e.g. 687.2 ng/mL in Furo [26] and 570.9 ng/mL in Warrington, et al. [20]) with the unadulterated urine samples from those were on buprenorphine/naloxone. This similarity should be explored further in relation to the implication of pharmacokinetics and pharmacodynamics of naloxone in the system.

#### **4.3 Clinical applications**

The results of this study can be used to help clinicians interpret urine toxicology test results more accurately. In this section, seven simulated cases based on previously encountered results are discussed to present how to apply the findings of this study to the daily practice of OUD treatment. The cases and their associated laboratory values as well as the buprenorphine prescription dosage are summarized in **Table 4**.

#### *4.3.1 Case 1*

[Bup 278 ng/mL, Norbup 635 ng/mL, Bup/Norbup 0.44, Nal 687 ng/mL, Cre 133.9 mg/dL, Bup/Nal 16 mg/4 mg per day, appropriate case].

This is a typical pattern of urine toxicology results. If patients are taking "buprenorphine" daily, "norbuprenorphine" is usually higher than "buprenorphine", [16] and creatinine is within normal range (20-400 mg/dL) (The ARUP laboratory criteria). Therefore, this is an appropriate urine toxicology case for those who are compliant with buprenorphine/naloxone treatment.

#### *4.3.2 Case 2*

[Bup >2000 ng/mL, Norbup 17 ng/mL, Bup/Norbup 117.65, Nal >2000 ng/mL, Cre 97.2 mg/dL, Bup/Nal 16 mg/4 mg per day, inappropriate case].


*Bup = Buprenorphine, Norbup = Norbuprenorphine, Nal = Naloxone, Cre = Creatinine. The units are listed in the top column. The level > 2000 ng/mL was considered 2000 ng/mL for the calculations.*

#### **Table 4.**

*7 simulated cases in clinical practice and their associated laboratory values.*

This is a typical pattern of urine adulteration of having dipped buprenorphine/ naloxone film directly into the urine sample. The ratio between buprenorphine/norbuprenorphine is >50 [17]. The very high naloxone level (>2000 ng/mL) confirms this suspicion of adulteration, consistent with the results of this study.

#### *4.3.3 Case 3*

[Bup >2000 ng/mL, Norbup >2000 ng/mL, Bup/Norbup 1.0, Nal >2000 ng/mL, Cre 306.7 mg, Bup/Nal 16 mg/4 mg per day, appropriate case].

This case has a high level of naloxone >2000 ng/mL, so there is suspicion of urine adulteration; however, this is an example, in which a high naloxone level does not mean urine adulteration. As Furo's study found that 15% of urine samples had "naloxone" levels >2000 ng/mL [26], a high level of naloxone can also be due to other reasons. This case illustrates that we can use a high "naloxone" level to confirm urine adulteration only if the buprenorphine/naloxone ratio is >50:1; "naloxone" level itself cannot be the main determining factor of urine adulteration. In other words, the judgment of urine adulteration should be based on high buprenorphine/norbuprenorphine ratio. Therefore, this is an appropriate case of buprenorphine/naloxone treatment. One might wonder why is this patient's "naloxone" level so high? The answer is that this patient might be dehydrated at the time of this urine collection, indicated by the high creatinine level, so the buprenorphine, norbuprenorphine, naloxone and creatinine are all consequently very high. If this patient were well hydrated, the results should have been similar to Case 1 with a much lower creatinine level. Thus, this example should be appropriate for the buprenorphine/norbuprenorphine treatment.

#### *4.3.4 Case 4*

[Bup >2000 ng/mL, Norbup 10 ng/mL, Bup/Norbup 200, Nal <2 ng/mL, Cre 82.6 mg/dL, Bup 16 mg per day, inappropriate case].

This case is an adulteration case in which the patient is prescribed buprenorphine only medication. This patient has not taken this medication for a while, indicated by the low norbuprenorphine level. This case has a high ratio of buprenorphine/norbuprenorphine, but naloxone is undetectable, which means that prior to the urine collection, buprenorphine medication has been crushed into powder and dissolved into the sample. As a result, the ratio between buprenorphine/norbuprenorphine is high (>50), but there is undetectable naloxone level (<2 ng/mL). Undetectable naloxone level should be always suspected with buprenorphine monotherapy.

#### *4.3.5 Case 5*

[Bup >2000 ng/mL, Norbup 17 ng/mL, Bup/Norbup 117.65, Nal 5 ng/mL, Cre 179.2 mg/dL, Bup/Nal 16 mg/4 mg per day, inappropriate case].

This is another case of urine adulteration as indicated with a high buprenorphine/ norbuprenorphine ratio (>50:1). However, "naloxone" is detected at a very small amount. Because of the high buprenorphine/norbuprenorphine ratio, we expect a high level of "naloxone" if buprenorphine/naloxone was dipped in this urine sample. Otherwise, we expect undetectable level of "naloxone" if buprenorphine only medication was dissolved into the urine sample. The small amount of "naloxone's being detected means that this patient probably has taken buprenorphine/naloxone at least 24 hours before this urine collection [15], and thus there was a small amount of

#### *Detecting Naloxone in Adulterated Urine Samples: Can Naloxone Be Detected When… DOI: http://dx.doi.org/10.5772/intechopen.109412*

"naloxone" residue detected. Compared with buprenorphine, the naloxone's half-life is much shorter, and thus, a low level of naloxone can be detected while relatively higher level of buprenorphine and norbuprenorphine still remained in the system after an intermittent use of buprenorphine/naloxone. In addition, this patient tampered the urine sample with buprenorphine only medication, which is indicated by the high buprenorphine/norbuprenorphine ratio. If buprenorphine/naloxone combined medication was dipped into this urine sample, the naloxone level would have been much higher as indicated by the results of this study, unless the patient has some issues of naloxone metabolism such as the enzyme to metabolize naloxone is inhibited by certain medication(s) or genetically. This patient is prescribed with buprenorphine/naloxone combined medication, so it is uncertain as to why this patient added additional buprenorphine to the urine.

#### *4.3.6 Case 6*

[Bup >2000 ng/mL, Norbup 2000 ng/mL, Bup/Norbup 1.0, Nal 2000 ng/mL, Cre 82.5 mg/dL, Bup/Nal 16 mg/4 mg per day, appropriate case].

This case has high "buprenorphine", "norbuprenorphine" and "naloxone" levels, so we can speculate dehydration; however, "creatinine" level is not as high as Case 3, and therefore, we can rule out dehydration. The ratio between buprenorphine to norbuprenorphine is <50:1, so it is unlikely that this sample is adulterated. This might be a case with high metabolism patient, which causes high levels of all components. This is rare but can happen. Thus, this is another example of a high naloxone level, but it does not mean urine adulteration.

#### *4.3.7 Case 7*

[Bup 286 ng/mL, Norbup 569 ng/mL, Bup/Norbup o.5, Nal 71 ng/mL, Cre 67.5 mg/ dL, Bup 16 mg per day due to naloxone allergies, inappropriate case].

This patient has an appropriate "buprenorphine" to "norbuprenorphine" ratio (<50:1) with low level of "naloxone"; however, this result is not appropriate because this patient was on buprenorphine monotherapy due to naloxone allergies. The detected "naloxone" suggests that she has been taking buprenorphine/naloxone despite her being prescribed buprenorphine monotherapy. Buprenorphine only product has a higher street value than buprenorphine/norbuprenorphine combined medication by approximately 20% [27]. Therefore, some patients might trade their buprenorphine with buprenorphine/naloxone combination medication for the marginal profit.

In summary, as this study found, a high concentration of "naloxone" can confirm suspected urine adulteration if buprenorphine/norbuprenorphine ratio is >50:1. However, a high level of "naloxone" alone does not necessarily involve urine adulteration, especially without a high ratio of buprenorphine/norbuprenorphine. The patient might have dehydrated at the time of urine collection, or the patient might be a fast metabolizer of buprenorphine/naloxone. Therefore, we should monitor creatinine level because it can indicate the hydration status of the patient [28], as dehydration can cause high levels of "naloxone" in addition to high "buprenorphine" and "norbuprenorphine" levels. Furthermore, a patient with high metabolism can cause high levels of "buprenorphine", "norbuprenorphine" and "naloxone" without a corresponding high "creatinine" level. Finally, if "naloxone" is observed in urine sample, we can suspect that the patient is taking or had taken buprenorphine/naloxone

combined medication, while if no naloxone is identified, the patient is on buprenorphine monotherapy.

#### **4.4 Clinical implications**

The results of this study have implications on clinical practice in the care of OUD patients. With the increased use of unobserved urine toxicology collection in practice [29], the appropriate interpretation of urine drug tests remains a challenging but vital component of comprehensive patient care. Results from this study may suggest that the "naloxone" detected in adulterated urine samples may be dissolved "naloxone" instead of metabolized naloxone, or a fraction of naloxone at least. There are also healthcare policy implications with the controversy of mono-product formulation use in the United States as a harm reduction approach in the treatment of OUD [9]. Buprenorphine mono-product is used widely in other areas of the world [30] and has been suggested as a medication treatment option for patients struggling with adverse effects, thought to be potentially from naloxone absorption from sublingual buprenorphine/naloxone combined medication [9]. With increasing research findings that raise the concern of using "naloxone" levels in urine as an indicator of absorption of naloxone and potentially the cause of patient-reported adverse effects, these findings reinforce the recommendation for clinicians to also consider possible urine adulteration, resulting in elevated "naloxone" levels.

A high level of "naloxone" itself should trigger further assessment of the patient buprenorphine/naloxone regimen; however, by itself there is insufficient evidence to guide care. This information should be synthesized with other available urine toxicology screen parameters (buprenorphine, norbuprenorphine, creatinine and buprenorphine/norbuprenorphine ratio) to better inform the clinical picture. Moreover, the continuation of buprenorphine as a medication for OUD yields more benefits than risks in the public health approach of addressing opioid overdose mortality. Urine toxicology is only one piece of information to be used in a clinical evaluation. The overall goal of improvement of multiple biopsychosocial domains and patient-centered outcomes remains the driving force in clinical decision-making.

Finally, the results of this study might support the stipulation that sublingual naloxone can be minimally absorbed and metabolized in the system and thus exerts insignificant effects clinically when taken sublingually, which might consequently contribute to the controversy on buprenorphine/naloxone combined product versus buprenorphine monotherapy in the buprenorphine induction process [31, 32]. The results of this study would give us an insight on these controversial issues.

#### **4.5 Limitations**

The limitations of this study include the small sample size and reliance on subjects' report of substance and medication use. Findings from this study may be used to apply for funding to support a larger sample size study and additional substance testing of all samples. While the potential effects of unreported substance use are unknown, the overall trend in this study's results is unlikely to be impacted. An additional limitation is that this study evaluated the addition of varying buprenorphine/ naloxone levels via film into water and urine samples as a proxy for adulteration. The actual adulteration techniques used by individuals may vary widely. A clinical trial evaluating "naloxone" levels in adulterated and non-adulterated urine samples of

*Detecting Naloxone in Adulterated Urine Samples: Can Naloxone Be Detected When… DOI: http://dx.doi.org/10.5772/intechopen.109412*

patients prescribed buprenorphine/naloxone would be difficult in nature. To address the range in adulteration techniques, we used multiple categories of surface areas, dipping duration, temperatures, and concentrations of both urine and water samples to assimilate a variety of methods and observe the resulting "naloxone" levels. Finally, we also acknowledge that some clinicians use urine toxicology that do not report "naloxone" levels in certain clinic practices, and therefore these findings may not be clinically useful to all providers.

#### **5. Conclusions**

The urine "naloxone" level by dissolving buprenorphine/naloxone film is very sensitive to the area and the time that the film come into contact with the urine samples. Attempted adulteration of the urine sample is likely yield supratherapeutic levels of "naloxone". At this time, there is no consensus as to what level to set the "naloxone" concentration as a parameter to determine the legitimacy of patient urine samples except some research reports. While "naloxone" level is a simple component to test for in resource-challenged practices, other metabolites such as "buprenorphine" and its metabolite levels should be examined to guide clinical decisions. In summary, the results of this study have provided a further insight into interpreting urine drug screening test results for OUD patients with buprenorphine treatment. Strict monitoring of urine toxicology is by no means a punitive process but to improve the outcome of OUD treatment. Future study can focus on differentiating naloxone molecules that were dissolved "naloxone" versus those that were renally excreted, which can enhance our understanding on the pharmacokinetics and pharmacodynamics of naloxone.

#### **Acknowledgements**

We would like to express our sincere and deep appreciation for the editors and reviewers who provided us with valuable feedback. We would also thank Editage for editing the final version of this manuscript.

#### **Authors' contributions**

H.F. carried out the research experiment and wrote the first draft of the paper excerpt discussion and conclusion sections. T.H. wrote the discussion and conclusion sections and completed the manuscript with support with S.A. who edited the draft form of this manuscript. Y.Z. completed the manuscript by editing the final version.

#### **Funding**

Not applicable.

#### **Competing interests**

There is no conflict of interests among the authors.

### **Consent for publication**

All authors agree with publishing this manuscript.

#### **Note**

Correspondence concerning this article should be addressed to Hiroko Furo (furo@uthscsa.edu), Department of Psychiatry and Behavioral Sciences, the University of Texas Health at San Antonio, San Antonio, TX, USA.

### **Ethics approval and consent to participate**

The Institutional Review Board (IRB) application to the University of Texas Health at San Antonio was approved (IRB Protocol ID: 20220593HU), and the written consents were obtained by all participants.

### **Availability of data and materials**

The datasets produced for the data analysis for this study are not publicly available due to the confidentiality of participants but are available in a de-identified form from the corresponding author on request.

### **Abbreviations**


*Detecting Naloxone in Adulterated Urine Samples: Can Naloxone Be Detected When… DOI: http://dx.doi.org/10.5772/intechopen.109412*

### **Author details**

Hiroko Furo1,2,3,4\*, Tony Lin4 , Yi Yuan Zhou2 and Sarah Abdelsayed4

1 Department of Psychiatry and Behavioral Sciences, The University of Texas Health at San Antonio, San Antonio, TX, USA

2 Department of Pathology, The University of Texas Health at San Antonio, San Antonio, TX, USA

3 Department of Biomedical Informatics, State University of New York (SUNY) at Buffalo, Buffalo, NY, USA

4 Department of Family Medicine, State University of New York (SUNY) at Buffalo, Buffalo, NY, USA

\*Address all correspondence to: hfuro1@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] "Naloxone". Drugbank Online. 11/28/22. https://go.drugbank.com/ drugs/DB01183

[2] Koyyalagunta D. Opioid analgesics. In: Pain Management. Netherlands: Elsevier; 2007. pp. 939-964

[3] Jordan MR, Morrisonponce D. Naloxone. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022. Available from: https://www.ncbi.nlm.nih.gov/books/ NBK441910/ . [Accessed: Jul 11, 2022]

[4] Nasser AF, Heidbreder C, Liu Y, et al. Pharmacokinetics of sublingual buprenorphine and naloxone in subjects with mild to severe hepatic impairment (child-pugh classes A, B, and C), in hepatitis C virus-seropositive subjects, and in healthy volunteers. Clinical Pharmacokinetics. 2015;**54**:837-849. DOI: 10.1007/s40262-015-0238-6

[5] Coe MA, Lofwall MR, Walsh SL. Buprenorphine pharmacology review: Update on transmucosal and longacting formulations. Journal of Addiction Medicine. 2019;**13**(2):93-103. DOI: 10.1097/ADM.0000000000000457

[6] Available from: https:// www.fda.gov/media/100429/ download#:~:text=Naloxone%20 is%20absorbed%20not%20only,is%20 within%201%2D2%20minutes

[7] Preston KL, Bigelow GE, Liebson IA. Effects of sublingually given naloxone in opioid-dependent human volunteers. Drug and Alcohol Dependence. 1990;*25*(1):27-34. DOI: 10.1016/0376-8716(90)90136-3

[8] NIDA. Naloxone DrugFacts. National Institute on Drug Abuse website.

2022 Available from: https://nida.nih. gov/publications/drugfacts/naloxone [Accessed: November 26, 2022]

[9] Blazes CK, Morrow JD. Reconsidering the usefulness of adding naloxone to buprenorphine. Frontiers in Psychiatry. 2020;**11**:549272. DOI: 10.3389/ fpsyt.2020.549272

[10] Mendelson J, Jones RT, Welm S, et al. Buprenorphine and naloxone combinations: the effects of three dose ratios in morphine-stabilized, opiate-dependent volunteers. Psychopharmacology. 1999;**141**(1):37-46. DOI: 10.1007/s002130050804

[11] Tzatzarakis MN, Vakonaki E, Kovatsi L, et al. Determination of buprenorphine, norbuprenorphine and naloxone in fingernail clippings and urine of patients under opioid substitution therapy. Journal of Analytical Toxicology. 2015;**39**(4):313-320

[12] Stone, JA Pesce AJ, Fitzgerald RL. 2017 Fake news, alternative facts or just normal pharmacokinetics? High urine naloxone concentrations in patients prescribed sublingual buprenorphine– naloxone (BNX). MSACL EU Abstract

[13] Strickland DM, Burson JK. Sublingual absorption of naloxone in a large clinical population. Journal of Drug Metabolic Toxicology. 2018;**09**(02):1-4

[14] Rosado J, Walsh SL, Bigelow GE, Strain EC. Sublingual buprenorphine/ naloxone precipitated withdrawal in subjects maintained on 100mg of daily methadone. Drug and Alcohol Dependence. 2007;**90**(2-3):261-269. DOI: 10.1016/j.drugalcdep.2007.04.006

[15] Heikman P, Häkkinen M, Gergov M, Ojanperä I. Urine naloxone concentration *Detecting Naloxone in Adulterated Urine Samples: Can Naloxone Be Detected When… DOI: http://dx.doi.org/10.5772/intechopen.109412*

at different phases of buprenorphine maintenance treatment: Urine naloxone concentration at buprenorphine maintenance treatment. Drug Test Analysis. 2014;**6**(3):220-225

[16] Furo H, Schwartz DG, Sullivan RW, Elkin PL. Buprenorphine dosage and urine quantitative buprenorphine, norbuprenorphine, and creatinine levels in an office-based opioid treatment program. Substance Abuse. 2021;**15**:1-9 11782218211061749. DOI: 10.1177/11782218211061749

[17] Accurso AJ, Lee JD, McNeely J. High prevalence of urine tampering in an office-based opioid treatment practice detected by evaluating the norbuprenorphine to buprenorphine ratio. Journal of Substance Abuse Treatment. 2017;**83**:62-67. DOI: 10.1016/j. jsat.2017.10.002

[18] Donroe JH, Holt SR, O'Connor PG, Sukumar N, Tetrault JM. Interpreting quantitative urine buprenorphine and norbuprenorphine levels in officebased clinical practice. Drug and Alcohol Dependence. 2017;**180**:46-51. DOI: 10.1016/j.drugalcdep.2017.07.040

[19] Suzuki J, Zinser J, Issa M, Rodriguez C. Quantitative testing of buprenorphine and norbuprenorphine to identify urine sample spiking during office-based opioid treatment. Substance Abuse. 2017;**38**(4):504-507. DOI: 10.1080/08897077.2017.1356796

[20] Warrington JS, Booth K, Warrington GS, et al. Use of urinary naloxone levels in a single provider practice: a case study. Addiction Science & Clinical Practice. 2020;**15**:3. DOI: 10.1186/s13722-020-0178-9

[21] Burns MM, Law TC, Rifai N, Shannon MW. Naloxone produces a positive urine opiate screen. Pediatric Research. 1999;**45**(7):80-80

[22] Clarke SFJ, Dargan PI, Jones AL. Naloxone in opioid poisoning: Walking the tightrope. Emergency Medicine Journal. 2005;**22**:612-616

[23] Available from: https://www.ibm. com/spss

[24] "Buprenorphine". Drug enforcement administration—diversion control division: Drug and chemical evaluation section. 11/28/22. Available from: https://www.deadiversion.usdoj.gov/ drug\_chem\_info/buprenorphine.pdf

[25] Walsh L. Buprenorphine. Available from: https://www.samhsa.gov/ medication-assisted-treatment/ treatment/buprenorphine,

[26] "Interpretation of urine drug test results of buprenorphine, norbuprenorphine, naloxone, creatinine levels in buprenorphine treatment program. Work-in-Progress. Furo H.

[27] Hswen Y, Zhang A, Brownstein JS. Leveraging black-market street buprenorphine pricing to increase capacity to treat opioid addiction, 2010-2018. Preventive Medicine. 2020;**137**:106105. DOI: 10.1016/j. ypmed.2020.106105

[28] Weigand TJ, editor. Use and interpretation of buprenorphine metabolite profiles during maintenance treatment. In: Poster Presented at: 47th American Society of Addiction Medicine (ASAM) Annual Conference, April 14-17. MD: Baltimore; 2016

[29] Rao PN, Jotwani R, Joshi J, Gulati A, Mehta N. Reevaluating chronic opioid monitoring during and after the COVID-19 pandemic. Pain Management. 2020;**10**(6):353-358. DOI: 10.2217/ pmt-2020-0063

[30] Yokell MA, Zaller ND, Green TC, Rich JD. Buprenorphine and

buprenorphine/naloxone diversion, misuse, and illicit use: an international review. Current Drug Abuse Reviews. 2011;**4**(1):28-41. DOI: 10.2174/1874473711104010028

[31] Rzasa Lynn R, Galinkin JL. Naloxone dosage for opioid reversal: current evidence and clinical implications. Therory of Advanced Drug Safety. 2018;**9**(1):63-88. DOI: 10.1177/2042098617744161

[32] Dooley J, Gerber-Finn L, Antone I, Guilfoyle J, Blakelock B, Balfour-Boehm J, et al. Buprenorphinenaloxone use in pregnancy for treatment of opioid dependence: Retrospective cohort study of 30 patients. Canadian Family Physician. 2016;**62**(4):e194-e200

### **Chapter 7**

## Usefulness of Urine Tests in the Prevention, Diagnosis, Treatment and Prognosis of Pathologies Present during Pregnancy

*Noren Villalobos*

#### **Abstract**

Pregnancy produces physiological changes in the woman necessary to be able to bring it to a happy term. However, they can favor the development of pathologies in various organs and systems, ranging from urinary infections, diabetes mellitus or gestational to hypertensive disorders of pregnancy. Which produce substances that are excreted through the urine. There is also excretion of metabolites which can be evaluated for the diagnosis and prognosis of certain chromosomopathies. These substances, when measured or quantified, provide bases for diagnosis, prevention, and allow decisions to be made regarding timely treatment in many of them.

**Keywords:** urinalysis, pregnancy, pathologies of pregnancy, diagnosis, urinary tract infections

#### **1. Introduction**

Pregnancy (EMB) is considered a physiological state where the maternal organism must adapt to the allograft it carries in its body [1]. To do this, it undergoes a series of modifications or physiological changes that allow it to adapt to the EMB in a continuous and dynamic way, which occur at the level of all organs and systems, and in turn are influenced by multiple factors that include the age of the woman, previous pregnancies, physical and nutritional status, presence of previous diseases or pathologies, among others [2]. These changes regress after the birth of the fetus in a period of approximately 40 days or until the moment the menstrual cycle resumes, returning the mother to the state prior to pregnancy and preparing her body for a new pregnancy [1, 2].

These changes known as gravid or physiological modifications of pregnancy (MFE) can be monitored through laboratory tests and diagnostic aid images (such as ultrasound) which can be routinely performed quarterly in normal EMB or vary their frequency in those cases in which complications or pathologies associated with it appear, which are capable of modifying its course or aggravating or unmasking a pre-existing problem [3].

#### **1.1 MFEs comprise**


The kidney is one of the organs that undergo important changes with the EMB which begin from the 6th week beginning with anatomical changes that include dilation of the pelvis and renal calyces and ureters, all this under the action of pregnancy hormones. Progesterone and estrogens [1, 2]. This causes an increase in urinary dead space, at the same time that renal vascularization increases with an increase in interstitial volume, which in turn causes an increase in kidney length of approximately 1 to 1.5 cm. compared to a normal adult (4.5). The kidney recovers its normal size approximately 6 months postpartum, even when the puerperium has already ended.

All of this leads to changes in renal physiology from the hemodynamic point of view, glomerular filtration, water and electrolyte management, and changes in the renin-angiotensin-aldosterone system [4].

A predisposition to the appearance of urinary tract infections (UI) occurs due to compression of the right ureter by dextrorotation of the uterus, to which the sigmoid colon contributes, which compresses the right ureter, and to the decrease in its peristalsis due to the action of progesterone, which favors the appearance of hydronephrosis. in the right kidney, slowing of urine transit through the ureter causing retrograde stasis. Contributing to this is the fact of the residual presence of urine in the urinary bladder, with a decrease in its tone, and a relative increase in its capacity as the EMB progresses due to uterine compression, which even produces reflux into the ureter, and may be up to be for 24 hours or more without being eliminated properly. Its mucosa becomes hyperemic and edematous, making it susceptible to infections and trauma [4].

Renal blood flow increases between 35 and 60%, therefore glomerular filtration increases between 50 and 60% together with the reabsorption of water and electrolytes to maintain the hydroelectrolytic balance [4–6].

During EMB, amino acids and water-soluble vitamins are lost in greater amounts than in non-EMB. Creatinine and uric acid decrease so that normal values for pregnant women are considered normal at 0.8 mgrs.%, and a greater increase is considered suspicious of kidney disease. Creatinine clearance increases by 30%. therefore, values less than 1 37 ml must be evaluated. Glycosuria is not necessarily abnormal and there may be non-significant proteinuria of 115 to 260 mg/day [1].

#### **2. Utilization of urinalysis during pregnancy**

#### **2.1 Urinary tract infections**

The appearance of urinary tract infections (UTI) is possibly the most frequent pathology during EMB, with a prevalence ranging between 14 and 48% [7], favored by the MFE that occur in the urinary tract and the alkalinization of the urinary tract. Urine [8, 9]. It is associated with other complications of EMB including pyelonephritis, preterm delivery, low birth weight newborns, and increased perinatal mortality [10, 11].

Preterm delivery (PP) is the main complication associated with UTIs, since bacterial infection in maternal and fetal tissues causes the release of endotoxins and exotoxins, cytokines, tumor necrosis factor, interleukin-1 beta, interleukins 6 and 8, granulocyte colony-stimulating factor and other factors, which stimulate the production and release of prostaglandins, leading to uterine contractions and neutrophil activity, which in turn stimulate the synthesis and production of metalloproteinases, causing membrane rupture and collagen remodeling of the cervix [12].

UTI is defined as the presence of 100,000 germs per cubic centimeter (cc) of urine in asymptomatic patients, or greater than 100,000 germs per cc of urine and leukocytes greater than 7 leukocytes per cc of urine in a symptomatic patient [10]. In most cases, it begins in the urinary bladder, favored by MGE, which in turn will allow it to ascend to the kidney [7].

#### *2.1.1 Types of urinary tract infection*

Asymptomatic bacteriuria: It is the presence of 100,000 organisms per milliliter (ml) in 2 consecutive cultures with the absence of symptoms, constituting a 40% risk factor for acute cystitis and 25 to 30% for pyelonephritis in pregnancy [9].

UTI: Presence of 100,000 organisms per ml of urine in asymptomatic patients or more than 100,000 accompanied by more than 7 cells in a symptomatic patient [9].

Cystitis: It constitutes the lower UTI characterized by inflammation of the urinary bladder due to bacterial causes or not (radiation) or viral. It occurs in 1 to 2% of pregnant women, being negative in 60% in the first screenings. It is complicated between 15 to 50% with pyelonephritis [9].

Pyelonephritis: upper UTI occurs in approximately 0.5 to 2% of all pregnancies [7, 9].

Now, what should we study in the urine test to help us diagnose UTIs or other pathologies.

#### **2.2 Urinary sediment**

The urinary sediment is obtained from a urine sample centrifuged at 2000 revolutions per minute, from which different types of cells originating from the urinary tract epithelium, or other cells such as leukocytes, red blood cells, platelets, renal cells, hyaline casts, are obtained. Leukocyte casts, amorphous phosphates, calcium crystals, uric acid, urates, lipid droplets, cholesterol crystals, proteins [13].

The presence of organisms such as bacteria, which can be typical of the urinary tract or contamination of the vagina, is frequent, as well as protozoa (vaginal trichomonas) and fungi such as candida vaginalis, as well as menstrual products and even

spermatozoa [13]. Always contamination vaginal is accompanied by leukocytes and vaginal epithelial cells, so it is necessary to examine the patient's vagina with a speculum when we have these results. Gardnerella vaginalis is another germ that causes infections and vaginal discharges that easily contaminate the urine test.

In the urine analysis, the presence of leucocyte esterase can be evaluated, which is an enzyme secreted by neutrophils, constituting a marker of infection [14, 15]. It has a sensitivity of 83% and a specificity of 78% [16].

Another test used is the nitrite test. It is based on the reduction of nitrates to nitrites carried out by enterobacteria by action of the enzyme nitrite reductase, for which it is necessary that the urine be found without mobilizing for 4 hours, its specificity being that it does not react with other substances [17]. Its sensitivity is 53% and a specificity of 78% [16, 18].

When both studies are combined, they reach a sensitivity of 98% and a specificity of 95%, which makes them very useful in the diagnosis of urinary tract infections [17].

#### **2.3 Ph in urine**

The pH of the urine covers the limits of acidity and alkalinity from 5.0 to 8.8. Ph greater than or equal to 6.0 is considered altered [17]. Its determination is a reflection of the buffered ion concentration and not a net measure of acids [18].

Acidic urine, with a pH less than 4.5, may be a reflection of metabolic acidosis, such as diabetic ketoacidosis, which can affect a patient with poorly controlled gestational diabetes or type I or II diabetes during pregnancy. Cases of patients with chronic diarrhea, a diet rich in meat, or in cases of chronic respiratory failure [18].

In contrast, alkaline urine presents with a pH greater than 8.0 and may be due to renal tubular acidosis, metabolic alkalosis which may be caused by pregnancy vomiting in the first trimester or in the case of hyperemesis gravidarum, or in the case administration of diuretics, in respiratory alkalosis or in cases of urinary infections by urease-producing germs such as Proteus miriabilis or in cases of a vegetarian diet [18].

In patients with alkanuria an alkaline pH is reported, which occurs in the presence of bacteria, urinary infection or diets rich in citrus or vegetables or the presence of certain drugs. It is also the product of the presence of lithiasis due to calcium carbonate, calcium phosphate and magnesium phosphate. However, the presence of acid urine is identified with aciduria, the product of respiratory or metabolic acidosis. When there is tubular acidosis, the urine is alkaline and the blood pH shows acidity [19].

During the pregnancy motivated by the MFE, changes in the breathing of the pregnant woman begin to appear from the eighth week, they begin with hyperventilation and a slight dyspnea which increases gradually throughout the pregnancy due to anatomical modifications by increasing the internal vertical diameters. and circumference of the ribcage when the uterus increases in size and volume, which in turn compresses the abdominal viscera against the diaphragm, thereby limiting its mobility, thereby increasing intra-abdominal pressure, which has its impact at the of lung volumes. This in turn causes the reduction of PCO2 to 30 mmHg by the action of progesterone, while increasing PO2 to 107 mmHg. Serum bicarbonate decreases to 20 mEq/L. By increasing renal excretion, it modifies the pH from 0.02 to 0.06 as a metabolic compensation for respiratory alkalosis (4.5).

*Usefulness of Urine Tests in the Prevention, Diagnosis, Treatment and Prognosis of Pathologies… DOI: http://dx.doi.org/10.5772/intechopen.109540*

All of this has as a consequence that the urine tends to become alkaline with a Ph of 6.0 or more, which, together with the gravid changes that affect the urinary system, will favor the appearance of UTI.

#### **2.4 Hormonal study in a urine sample**

Human chorionic gonatropin (HCG) is a protein synthesized by embryonic tissues, made up of 2 amino acid chains called alpha and beta. Its secretion is related to the growth of trophoblastic tissue during pregnancy, reaching its maximum levels between weeks 3 and 9 of the same [20].

Its concentration varies substantially in serum and urine [20]. For this reason, excretion in urine has allowed the development of pregnancy tests due to their levels in urine to make a diagnosis of it in a simple way, which can be carried out by patients without the need to go to a laboratory, but without specifying the conditions. Weeks of gestation, which is done by evaluating the plasmatic levels of its beta fraction.

It is considered responsible for the nausea and vomiting of pregnancy. Its presence, in addition to allowing the diagnosis of normal pregnancy, allows the diagnosis of ectopic pregnancy or gestational trophoblastic disease in any of its forms, which in most cases can occur with hypertensive disorders of pregnancy when its levels quadruple the values of the normal pregnancies. However, it can give false negatives when performed between weeks 41 and 109 of pregnancy, due to the decrease in trophoblastic tissue that produces it [20].

#### **2.5 Presence of metabolites in urine**

#### *2.5.1 Ketone bodies*

The presence of ketone bodies in urine during EMB may be related to the presence of diabetes mellitus. It must be known if this diabetes was type I or II and was present before it or if it is gestational diabetes that develops during it from the 24th week of pregnancy and which can disappear after it or the patient remains diabetic. Type II [21].

The MFE that leads to changes in energy distribution during pregnancy must be taken into account [21]. At the beginning of EMB, there is an increase in insulin sensitivity with a decrease in fasting plasma glucose levels and a slight decrease in hepatic glucose production. At the end of the first trimester and during the second trimester of gestation, insulin sensitivity decreases, finding its highest level in the third trimester, with a 30% increase in hepatic glucose secretion and a 40–50% decrease in glucose mediated by insulin, leading to decreased insulin sensitivity with a predisposition to accelerated fasting ketosis and increased fasting maternal glycemia and fatty acids [21].

Gestational diabetes is a common disorder that can affect pregnancy, capable of causing maternal and fetal complications such as neural tube malformations that include anencephaly, spina bifida, renal agenesis and hypoplasia, cardiac disorders such as tetralogy of Fallot, atrioventricular septal defects., coarctation of the aorta, ventricular septal defect, and musculoskeletal diseases and injuries such as agenesis of the sacrum, cleft palate, and high risk of preeclampsia, preterm delivery, fetal malformations, and cesarean sections due to the presence of fetal macrosomia [21–23].

Diabetic ketoacidosis in gestational diabetes has an incidence of 0.3 to 5%, so it is always necessary to rule out a history of type I and II diabetes, which are more easily and severely complicated than in non-pregnant patients [24].

#### *2.5.2 Proteinuria*

A separate chapter is constituted by proteinuria in pregnancy, which is extremely important for the diagnosis, evaluation, and prognosis of hypertensive disorders of pregnancy, preeclampsia-eclampsia, and its complications. It occurs in 2 to 10% of pregnancies [25] and is responsible for 26% of maternal deaths in Latin America and 9% in Africa and Asia.

Preeclampsia affects the endothelium systemically, proving a generalized endotheliosis that reaches all organs of the economy. In the kidney, at the level of the renal glomerulus, it produces glomeruloendotheliosis, which is manifested by proteinuria and oliguria that improves after fetal extraction [25].

It is necessary and important to know the classification of hypertensive disorders of pregnancy:


It is necessary to consider the presence of renal disease prior to pregnancy, which can cause proteinuria, for which it is necessary that it be present before the 20th week of gestation [27]. Its appearance after week 20 may constitute one of the first signs of the appearance of hypertensive disorders of pregnancy: preeclampsia [26].

For the diagnosis of proteinuria it is necessary to collect 24-hour urine with values equal to or greater than 300 mg/day [26].

In the presence of proteinuria, it is necessary to rule out different nephropathies ranging from chronic kidney disease in which a progression of kidney disease is considered [28], diabetes mellitus, human immunodeficiency virus (HIV) infection or autoimmune diseases such as Lupus. systemic erythematosus [29]. For this reason, any proteinuria greater than 300 mg/day needs to be evaluated and the respective differential diagnoses made, especially in the presence of autoimmune diseases.

#### *Usefulness of Urine Tests in the Prevention, Diagnosis, Treatment and Prognosis of Pathologies… DOI: http://dx.doi.org/10.5772/intechopen.109540*

Since preeclampsia is a cause of maternal morbidity and mortality in many countries [25], methods have been sought to simplify the diagnosis of proteinuria in pregnancy. One of the simplest is using Robert's reagent which produces protein precipitation forming a clear halo in a test tube which contains a urine sample. Densitometers have also been used to measure the increase in urinary density due to the increase in the proteins present in them. Its biggest disadvantage is that it can produce false positives, in which case it is necessary to resort to other tests to verify its results.

At present, test strips are used for a quick and simple diagnosis of proteinuria, easy to handle and apply both by primary health personnel in a prenatal consultation, and for use by the patient at home [30]. Although they can give results as false positives or negatives, motivated by the fact that the presence of pathologies prior to pregnancy or not must be known, it is useful in patients with blood pressures of 140/90 mmHg, for monitoring at home, which allows go to a care center if they are positive [30].

Another method that has been used to diagnose proteinuria in hypertensive disorders of pregnancy is the use of sulfosalicylic acid, looking for a fast, simple, economical and reproducible method for health personnel at any level. This chemical reagent is capable of producing protein precipitation through urine acidification. It has a sensitivity of 41.1% and a specificity of 97.7% [27].

Gestational proteinuria has been described in the absence of hypertensive disorders of pregnancy, which appears after 20 weeks of pregnancy. A careful evaluation of the same is necessary [31] motivated by the fact that previous pathologies must be ruled out, and take into account that it may be an early sign of preeclampsia [32].

Investigators have been looking for another method for the diagnosis of preeclampsia through urinalysis, which is why the presence of podocytes in urine or podocyturia has been described [33]. The podocyte is a cell that forms part of the basal epithelium of the glomerulus which is affected by renal pathologies, but it was thought that it was not altered in preeclampsia, but when the glomeruloendotheliosis of preeclampsia develops, these are affected. For this reason, the study of podocyturia as a diagnostic method for preeclampsia has been proposed. However, it is also present in cases of chronic arterial hypertension, diabetes mellitus and gestational diabetes, lupus nephritis, and chronic membranous nephropathy [30, 33]. For its study, synaptopodin is used as a marker, which is a protein that binds the actin of the podocytes with the cytoskeleton of the cells [33, 34].

#### *2.5.3 Other analyzable metabolites in urine samples*

Another method used for the diagnosis, evolution and prognosis of hypertensive disorders of pregnancy is the creatinine protein index. It is used as a simpler and faster option to perform proteinuria in 24 hours. It has a sensitivity of 90% and a specificity of 80% [35, 36].

Studies are underway to develop other methods for examining urine samples and using nuclear magnetic resonance techniques to diagnose the presence of specific metabolites or biomarkers for diagnosis of preeclampsia, gestational diabetes, preterm labor, and trisomy 21 [37].

With the appearance of the omics revolution, where through the analysis of metabolites and proteins of DNA and RNA in order to carry out diagnoses and treatments of different pathologies. These metabolomics search for low molecular weight cellular compounds such as carbohydrates, amino acids, peptides, nucleic acids, organic acids, vitamins, and lipids. They can be detected using nuclear magnetic

resonance spectrometry methods in samples of blood, urine, amniotic fluid, tissue secretions, and placental tissue. These data are evaluated through computer programs where they are analyzed, studied to give their interpretation and use these data in prevention, evolution, treatment and prognosis.

These studies can be carried out at the prenatal level, during the control itself, and they can be carried out through a urine sample [38].

Diaz et al. [39] in urine samples from the 14th week of pregnancy, searched for metabolic signatures using nuclear magnetic resonance metabolomin, which revealed specific urinary metabolic signatures for malformations of the central nervous system, trisomy 21, preterm delivery, diabetes pregnancy, intrauterine growth restriction and preeclampsia, demonstrating the value of the urinalysis profile as a complementary method of diagnosis and early prediction of various disorders that occur during pregnancy [39].

Cantowine et al. [40] found urinary concentrations of Bisphenaol A and Phosphate, a metabolite which are man-made products for industrial use in a wide variety of products ranging from the lining of canned food containers, water bottles and pipes water supply, products that are released in an innocuous way to the general population throughout the world, the general population being exposed to them.

It has been shown that BFA can affect the proliferative process of trophoblast cells through an estrogen receptor with an effect of apoptosis of trophoblast cells through tumor necrosis factors which act on the placenta and therefore on the development of preeclampsia [40].

Taking into account that preeclampsia occurs more frequently in primiparous women, the use of Fit-1 tyrosine kinase has been proposed, which constitutes a promising biomarker for preeclampsia obtained from a urine sample. It is a growth factor antagonist and vasoconstrictor and sensitizes the endothelium to respond to stress and endothelial dysfunction. The study is performed with nuclear magnetic resonance spectrophotometry. The Fit-1 measures moderate to severe placental dysfunction, knowing that the development of preeclampsia occurs due to inadequate placentation with abnormal development of placental vessels at the time of trophoblastic invasion, creating high-resistance vessels instead of low-resistance ones. Resistance that occurs in normal pregnancies. For this reason, it is capable of measuring the development of preeclampsia [41].

Other metabolites and biomarkers developed from a urine sample include increased excretion of amino acids as a result of kidney damage from preeclampsia. Choline increases in these patients while glycine decreases at the same time [42].

#### **3. Conclusions**

The physiological modifications of the pregnancy through its adaptive changes, favor the development of the pregnancy, keep the expectant mother in balance to achieve its completion with the best results. However, in turn they can contribute to the appearance of pathologies caused by the lack of adaptation or poor adaptation to it, such as hypertensive disorders of pregnancy, gestational diabetes, or UTI, among others. In the effort to prevent, diagnose, treat and have an adequate prognosis, it is sought to be able to make simple and reliable diagnoses and the evaluation of uranalysis is one of the simplest methods that we have. The sample is easy to obtain and its analysis ranges from the use of reactive tapes to the diagnosis of glycosuria, proteinuria or a UTI. Urinary sediment and Ph allow us complementary diagnoses

*Usefulness of Urine Tests in the Prevention, Diagnosis, Treatment and Prognosis of Pathologies… DOI: http://dx.doi.org/10.5772/intechopen.109540*

that make us suspect other pathologies such as pyelonephritis, gallbladder and renal lithiasis, facilitating their diagnosis. We must always clinically evaluate the patient according to the signs and symptoms they present in order to make the most assertive diagnosis possible. The presence of proteinuria should always make us suspect a hypertensive disorder of pregnancy, although its absence does not thus rule it out from the presence of renal pathologies or other systemic diseases present before pregnancy. The development of new techniques for evaluating a urine sample are the next stage in the diagnosis of pathologies through the metabolites excreted in the urine, which include the diagnosis of chromosomopathies, for which the urine test in pregnancy reaches every ever-greater importance.

#### **Conflict of interest**

"The authors declare no conflict of interest."

### **Author details**

Noren Villalobos1,2

1 High Risk Obstetric Service, Department of Obstetrics and Gynecology, University Hospital of Maracaibo, Dr. Armando Castillo Plaza Maternity, Maracaibo, Venezuela

2 Medicine School, School of Medicine, University of Zulia, Maracaibo, Venezuela

\*Address all correspondence to: norenvi@hotmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Tejada Pérez P, Cohen A, Font Arreaza IJ, Bermúdez C, Schuitemaker Requena JB. Modificaciones fisiológicas del embarazo e implicaciones farmacológicas: maternas, fetales y neonatales. Revista de Obstetricia y Ginecología de Venezuela. 2007;**67**(4):246-267

[2] Carrillo-Mora P, García-Franco A, Soto-Lara M, et al. Cambios fisiológicos durante el embarazo normal. Revista de la Facultad de Medicina de la UNAM. 2021;**64**(1):39-48. DOI: 10.22201/ fm.24484865e.2021.64.1.07

[3] Vinturache A, Khalil A, GLob. Maternal physiological changes in pregnancy in the continuous textbook, women's medicine series – obstetrics module. In: Khalil A, editor. Vol. 4. London: GLOWM; 2021. pp. 1-39

[4] Vinturache A, Khalil A, Global Library of Women's Medicine. DOI: 10.3843/glown.411323

[5] Purizaca M. Modificaciones fisiológicas en el embarazo. Rev Per Ginecol Obstet. 2010;**56**:57-69

[6] Carlin A, Alfirevic Z. Physiological changes of pregnancy and monitoring. Best Practice & Research Clinical Obstetrics and Gynaecology. 2008;**22**(5):801-823

[7] Ruiz-Rodríguez M,

Sánchez-Martínez Y, Suárez Cadena FC, García Ramírez JC. Prevalence and characterization of urinary tract infection in socially vulnerable pregnant women in Bucaramanga. Colombia. 2019;**69**(2):1-10. DOI: 10.15446/ revfacmed

[8] Mattuizzi A, Madar H, Froeliger A, Brun S, Sarrau M, Bardy C, et al. Infección urinaria y embarazo. EMC – Ginecología-Obstetricia. 2018;**54**(4):1-20

[9] Myers VC, Muntwiler E, Bill AH. The acid-base balance disturbance of pregnancy. The journal of biological chemestry. 1931;**98**(1):253-260

[10] Platte RO, Kim ED. Urinary Tract Infections in Pregnancy. Available from: https://emedicine.medscape.com/ article/452604-overview

[11] Werter DE, Kazemier BM, van Leeuwen E, et al. Diagnostic work-up of urinary tract infections in pregnancy: Study protocol of a prospective cohort study. BMJ Open. 2022;**12**:e063813. DOI: 10.1136/bmjopen-2022-063813

[12] Faneite P, Pérez Alonso MM, Sánchez WJ. In: González F, editor. Epidemiología y factores etiológicos. En Manejo del Parto Pretérmino. Caracas: Sociedad Venezolana de Obstetricia y Ginecología; 2012

[13] Fogazzi GB. The Urinary Sediment. An Integrated View. Third ed. Milano: Editorial Elsevier Srl; 2010. p. 251

[14] Ho ML, Liu WF, Tseng HY, Yu-Tzu Yeh YT, Tseng WT, et al. Quantitative determination of leukocyte esterase with a paper-based device. RSC Advances. 2020;**10**:27042. DOI: 10.1039/d0ra03306e

[15] Pfaller MA, Koontz FP. Laboratory evaluation of leukocyte esterase and nitrite tests for the detection of bacteriuria. Journal of clinical microbiology. 1985;**21**(5):840-842

[16] Estrada-Altamirano A, Figueroa-Damián R, Villagrana-Zesati R. Infección de vías urinarias en la mujer

*Usefulness of Urine Tests in the Prevention, Diagnosis, Treatment and Prognosis of Pathologies… DOI: http://dx.doi.org/10.5772/intechopen.109540*

embarazada. Importancia del escrutinio de bacteriuria asintomática durante la gestación. Perinatol Reprod Hum. 2010;**24**(3):182-186

[17] Myriam Ruiz-Rodríguez M, Sánchez-Martínez Y, Suárez-Cadena FC, García-Ramírez JC. Prevalence and characterization of urinary tract infection in socially vulnerable pregnant women in Bucaramanga, Colombia. Rev. Fac. Med. 2021;**69**(2):e77949. DOI: 10.15446/revfacmed.v69n2.77949

[18] Pagana K, Pagana T. Indicaciones e interpretación de resultados. In: Laboratorio Clínico. Primera edición en español ed. México: Editorial Manual moderno; 2014

[19] Prieto Valbuena JM, Yuste Ara JR. La clínica y el laboratorio. 23 edición ed. Barcelona: Elsevier editores; 2019. pp. 1160

[20] Velázquez N. La hormona gonadotrofina coriónica humana. Una molécula ubícua y versátil. Parte I. Rev Obstet Ginecol Venez. 2014;**74**(2):122-133

[21] Brajkovich I, Febres Balestrini F, Camejo M, Palacios A. Manual Venezolano De Diabetes Gestacional. Rev Venez Endocrinol Metab. 2016;**14**(1):56-90

[22] McMicking J, Lam AYR, et al, Global Library of women's Medicine, DOI: 10.3843/GLOWM.416413

[23] Vigil-De Gracia P, Olmedo J. Diabetes gestacional: conceptos actuales. Ginecología y Obstetricia de México. 2017;**85**(6):380-390

[24] Guevara-Valerio H, Mari-Zapata DD, Arévalo-Villa DI, Vargas-Aguilar DM, Etulain-González JE. Cetoacidosis diabética durante el embarazo: reporte de un caso. Ginecología y Obstetricia

de México. 2020;**88**(7):471-476. DOI: 10.24245/gom. V88i7.3547

[25] Pérez M, Pacheco M, Pérez K, Tineo N. Daño renal en pacientes preeclámpticas con criterios de gravedad. Revista de Obstetricia y Ginecología de Venezuela. 2020;**80**(3):176-186

[26] Kane SC, Wium L, et al, Global Library of women's Medicine. DOI: 10.3843/GLOWM.413253

[27] Velásquez Penagos JA, Zuleta Tobón JJ, López Jaramillo JD, Gómez Marulanda NL, Gómez Gallego J. Use of sulfosalicylic acid in the detection of proteinuria and its application to hypertensive problems in pregnancy. Iatreia. 2011;**24**(3):259-265

[28] Cravedi P, Remuzzi G. Pathophysiology of proteinuria and its value as an outcome measure in chronic kidney disease. British Journal of Clinical Pharmacology. 2013;**76**(4):516-523. DOI: 10.1111/bcp.12104

[29] Blom K, Odutayo A, Bramham K, Hladunewich MA. Pregnancy and glomerular disease a systematic review of the literature with management guidelines. Clinical Journal of the American Society of Nephrology. 2017;**12**:1862-1872. DOI: 10.2215/ CJN.00130117

[30] Jakubowski BE, Stevens R, Wilson H, Lavallee L, Brittain L, Crawford C, et al. Cross-sectional diagnostic accuracy study of self-testing for proteinuria during hypertensive pregnancies: The UDIP study. BJOG: An International Journal of Obstetrics and Gynaecology. 2022;**129**(13):2142-2148. DOI: 10.1111/1471-0528.17180

[31] Kattah A, Milic N, White W, Garovic V. Spot urine protein measurements in normotensive

pregnancies, pregnancies with isolated proteinuria and preeclampsia. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 2017;**313**:R418-R424. DOI: 10.1152/ ajpregu.00508.2016

[32] Morikawa M, Mayama M, Noshiro K, Saito Y, Nakagawa Akabane K, Umazume T, et al. Earlier onset of proteinuria or hypertension is a predictor of progression from gestational hypertension or gestational proteinuria to preeclampsia. Scientific Reports. 2021;**11**:12708. DOI: 10.1038/ s41598-021-92189-w

[33] Jim B, Jean-Louis P, Qipo A, Garry DA, Mian S, Matos T, et al. Podocyturia as a diagnostic marker for preeclampsia amongst highrisk pregnant patients. Journal of Pregnancy. 2012;**2012**:984630. DOI: 10.1155/2012/984630

[34] Ning L, Suleiman HY, Miner JH. Synaptopodin is dispensable for Normal Podocyte homeostasis but is protective in the context of acute Podocyte injury. JASN. 2020;**31**:2815-2832. DOI: 10.1681/ ASN.2020050572

[35] Díaz Colina NG, Chiroque Parra IR, García J, Villalobos Inciarte NE. Estudio comparativo entre la índice proteína/ creatinina en una muestra de orina al azar y proteinuria en 24 horas como método diagnóstico de preeclampsia. Revista de Obstetricia y Ginecología de Venezuela. 2022;**82**(1):59-66. DOI: 10.51288/00820108

[36] Pérez Dubuc KV, Vargas Torres PA, Gil Villegas Y, Vásquez Paredes LC. Trastornos hipertensivos del embarazo: relación del índice proteína/ creatinina en orina esporádica y proteinuria en 24 horas. Revista de Obstetricia y Ginecología de Venezuela. 2022;**82**(3):297-308. DOI: 10.51288/00820305

[37] Mayrink J, Leite DF, Nobrega GM, et al. Prediction of pregnancy-related hypertensive disorders using metabolomics: A systematic review. BMJ Open. 2022;**12**:e054697. DOI: 10.1136/ bmjopen-2021-054697

[38] Monni G, Atzori L, Corda V, Dessolis F, Iuculano A, Hurt KJ, et al. Metabolomics in prenatal medicine: A review. Frontiers in Medicine. 2021;**8**:645118. DOI: 10.3389/ fmed.2021.645118

[39] Diaz SO, Barros AS, Goodfellow BJ, et al. Second trimester maternal urine for the diagnosis of trisomy 21 and prediction of poor pregnancy outcomes. Journal of Proteome Research. 2013;**12**(6):2946-2957. DOI: 10.1021/ pr4002355

[40] Cantonwine DE, Meeker JD, Ferguson KK, Mukherjee B, Hauser R, McElrath TF. Urinary concentrations of Bisphenol a and phthalate metabolites measured during pregnancy and risk of preeclampsia. Environmental Health Perspectives. 2016;**124**(10):1651-1655. DOI: 10.1289/EHP188

[41] Austdal M, Skråstad RB, Gundersen AS, Austgulen R, Iversen A-C, et al. Metabolomic biomarkers in serum and urine in women with preeclampsia. PLoS One. 2014;**9**(3):e91923. DOI: 10.1371/journal. pone.0091923

[42] Austdal M, Brettas Silva G, Bowe S, Vestrheim Thomsen LC, Haugstad Tangerås L, Bjørge L, et al. Metabolomics identifies placental dysfunction and confirms Flt-1 (FMS-like tyrosine kinase receptor 1) biomarker specificity. Hypertension. 2019;**74**:1136-1143. DOI: 10.1161/ HYPERTENSIONAHA.119.13184

### *Edited by Tomasz Jarzembowski and Agnieszka Daca*

A urinalysis is a simple test that can help find urinary tract-related problems such as kidney disease. It can also pinpoint other serious problems not so closely related to kidneys, such as diabetes, liver disease, or even various cancers. Simply put, urine analyses may provide huge amounts of information to monitor a potential patient's condition. The history of analysis of urine for diagnostic purposes is quite long. It includes the detection of microbes as etiological agents of infection and the estimation of biochemical parameters such as glucose and protein concentration. Furthermore, the increase in the number of patients suffering from chronic kidney disease or other "civilization diseases" such as diabetes, hypertension, or obesity manifests the need for effective tools for specific and sensitive diagnosis. This book summarizes the state of the art in diagnosing infectious and non-infectious diseases based on urine analysis. Additionally, it focuses on novel techniques and applications used in everyday laboratory urinalysis. The history of analysis of urine for diagnostic purposes is quite long. It includes the detection of microbes as etiological agents of infection and the estimation of biochemical parameters such as glucose and protein concentration. Furthermore, the increase in the number of patients suffering from chronic kidney disease or other "civilization diseases" such as diabetes, hypertension, or obesity manifests the need for effective tools for specific and sensitive diagnosis. This book summarizes the state of the art in diagnosing infectious and non-infectious diseases based on urine analysis. Additionally, it focuses on novel techniques and applications used in everyday laboratory urinalysis.

Published in London, UK © 2024 IntechOpen © Michael Maasen / unsplash

Advances and Challenges in Urine Laboratory Analysis

Advances and Challenges in

Urine Laboratory Analysis

*Edited by Tomasz Jarzembowski and Agnieszka Daca*