Genetic polymorphisms detectable in human urine: their application to forensic individualization.

This review describes several types of genetic polymorphism, which have recently been identified in human urine in our laboratory, and have also been found in other human body fluids such as blood, saliva and semen. These include uropepsinogen, ribonuclease, deoxyribonuclease I (DNase I), deoxyribonuclease II (DNase II), 43-kDa glycoprotein, alpha-L-fucosidase, glutamate pyruvate transaminase, alpha-2-HS-glycoprotein, transferrin and vitamin D-binding protein. Several substances can be detected more easily in urine than in plasma. The concentrations of uropepsinogen, DNase I and DNase II in blood plasma are too low for analysis, whereas those in urine are high enough for easy typing. In practice, DNase I-polymorphism is one of the most useful genetic markers for practical purposes, because of its higher content in various body fluids including urine, a well-balanced gene frequency, and its easy and accurate detectability. Furthermore, several genetic markers previously identified in blood and/or other forensic samples can be phenotyped reproducibly and easily from the corresponding urine samples. Thus, urine, in addition to the convenience and non-invasive nature of its collection, is by no means inferior to blood as a sample source for typing in the field of forensic science. Biochemical and serological typing of genetic polymorphisms present in human urine could offer useful information to practising forensic biologists for forensic individualization of urine samples.

Electrophoretic analysis of a gastric cancer-associated acid proteinase using a highly sensitive detection system.

A highly sensitive detection system for acid proteinase separated on polyacrylamide gel was established. This system consisted of two-dimensional electrophoresis, combined with isoelectric focusing and polyacrylamide gel electrophoresis, and casein clotting (caseogram). Human urine, serum and gastric tissues obtained from normal individuals and gastric cancer patients were analyzed using this system. The previous electrophoretic method was not sufficiently sensitive to detected small amounts of pepsinogen (PG) C in normal urine. However, the new rapid and sensitive method clearly revealed its presence. In gastric tissue containing cancer cells, an additional proteinase, which was not present in normal tissue, was detected and named medium moving proteinase (MMP). MMP resembled PGs in alkaline stability rather than the non-PG proteinase, slow moving proteinase (SMP).

[The importance of the plasma proteins of the blood in maintaining the relative constancy of its hydrolytic properties]. / Znachenie belkov plazmy krovi v obespechenii otnositel'nogo postoianstva ee gidroliticheskikh svoistv.

Effects of hypervolemia, dehydration, activation and inhibition of urine-formation, i. v. administration of amylase and pepsinogen, experimental acute pancreatitis upon amylolytic activity of blood plasma, contents of pepsinogen in it, amylase and pepsinogen within the blood plasma protein factions, and excretion of enzymes with the urine, were studied in dogs. The data obtained suggest an important role of interconnection between amylase and pepsinogen with the plasma proteins in their renal and extrarenal excretion from the organism and in maintenance of a relative constancy of the contents and activity of enzymes in the blood. The affinity of the plasma proteins to their stains can indirectly characterise the transport capacity of the proteins in respect to amylase and pepsinogen.

High-performance liquid chromatography: purification and chromatographic behaviour of molecular variants of pepsinogen A from human urine.

By combining conventional DEAE chromatography with high-performance liquid chromatography on Sephacryl S-200 HR and Mono-Q columns, we have been able to isolate and fractionate human pepsinogen A (PGA) isozymogens from large amounts of urine. This method of fractionation is simple and allows one to obtain pepsinogen in a native non-denatured conformation. The isozymogens are homogeneous by electrophoretic and chromatographic criteria; this was confirmed by N-terminal amino acid sequencing. Purified PGA-3 and PGA-5 can be converted into an additional, more anionic, isoform on incubation at 37 degrees C. This isoform exists not only in vitro but also in vivo. The net negative charge of the PGA isozymogens is in the order PGA-5 less than deamidated PGA-5 less than PGA-3 less than deamidated PGA-3. Surprisingly, the elution order on the Mono-Q column was PGA-5/PGA-3/deamidated PGA-5/deamidated PGA-3. We have performed molecular modelling on PGA to investigate this phenomenon in terms of surface charge (not net charge) of the proteins. The model provides evidence that (1) only a fraction of the protein surface interacts with the support and (2) regions of localized charge at the protein surface may allow portions of the external surface to dominate chromatographic behaviour, resulting in a steering of the proteins with respect to the oppositely charged matrix. Pepsinogens may serve as model proteins for elucidating some of the variables that determine the chromatographic behaviour of proteins on ion-exchange columns.

Urinary pepsinogen I as a tumor marker of stomach cancer after total gastrectomy.

The possibility that urinary pepsinogen I is a tumor marker of stomach cancer after total gastrectomy was examined. To decide the cutoff level of urinary pepsinogen I after total gastrectomy, urine samples from 15 patients who had undergone total gastrectomy for stomach cancer in the early or advanced stages and had been free from recurrence for more than 5 years were examined by pepsinogen I-specific radioimmunoassay. The mean concentration of urinary pepsinogen I was 17.5 +/- 7.4 ng/ml and the cutoff level of urinary pepsinogen after total gastrectomy was set at 32 ng/ml (mean + 2 SD). Twenty-two of 74 cases who had undergone total gastrectomy for stomach cancer were regarded as positive. And 20 of these 22 positive cases had definite clinical signs of recurrence of stomach cancer. There were only two false-positive cases. These results suggest that urinary pepsinogen I will be an useful tumor marker in detecting the recurrence of stomach cancer after total gastrectomy.

The effect of acute and chronic protein loading on urinary pepsinogen A excretion.

In 8 healthy male volunteers, urinary excretion (UE) and fractional clearance (FC) of pepsinogen A (PGA), beta 2-microglobulin (beta 2-m) and albumin were measured after 6 days high protein diet (HPD; 2.0 g/kg/day) and compared to values obtained after 6 days low protein diet (LPD; 0.5 g/kg/day). In addition, the effect of an acute protein load (APL; 500 g beef) on these variables were measured. Both chronic and acute protein loading induced a rise in glomerular filtration rate (GFR) of about 10% together with a parallel rise in effective renal plasma flow. UE PGA and FC PGA increased both after HPD (UE PGA 1,707 +/- 1,106 ng/min; FC PGA 23 +/- 12%) as compared to LPD (UE PGA 1,200 +/- 987 ng/min, p less than 0.01; FC PGA 18 +/- 12%, p less than 0.05), and after APL (UE PGA 2,276 +/- 1,389 ng/min; FC PGA 26 +/- 16%) as compared to baseline (UE PGA 1,418 +/- 965 ng/min, p less than 0.02; FC PGA 21 +/- 12%, p less than 0.05). UE and FC of beta 2-m and albumin were not affected by protein loading. As PGA is nearly freely filtered, it is concluded that the increase in fractional PGA clearance reflects a decrease in fractional tubular PGA reabsorption. Our results suggest that an increase in fractional protein clearance after protein loading is not necessarily due to an impaired glomerular permselectivity but represents a decreased fractional tubular reabsorption as a result of a GFR-mediated increase in filtered load without a concomitant increase in tubular reabsorption.

Immunoblot technique to visualise serum pepsinogen A isozymogen patterns.

Pepsinogen A (PGA) isozymogen patterns in urine and gastric mucosa can be visualised in non-denatured polyacrylamide gel electrophoresis by showing proteolytic activity after the conversion of pepsinogen into pepsin by acid. This method is not suitable for visualising PGA patterns in serum due to low PGA concentrations. To obtain a more sensitive visualisation method an immunoblotting technique was developed. PGA isozymogen patterns from urine and sonified gastric mucosa specimens obtained by immunoblotting were identical with those obtained by activity staining. The immunostaining method was at least 50 times more sensitive. PGA isozymogen patterns could be visualised in serum. Preliminary results suggest that the PGA patterns in serum and gastric mucosa are identical. As an association has been found between the genetically determined PGA isozymogen patterns in gastric mucosa and gastric malignancies in man, immunoblotting of PGA isozymogens in serum may provide a screening tool for subjects at risk of malignant gastric disease.

Characterization of urinary peptic activity after total gastrectomy.

Urinary peptic activity, which has always been thought to originate from only the gastric mucosa, was detected in 26 of 50 totally gastrectomized patients with a mean value of 167 +/- 30 ng/ml. The gel filtration analysis suggested that the peptic activity of urine after total gastrectomy was detected at the same fractions as those of pepsinogens. Moreover, electrophoretical analysis of urine after total gastrectomy showed that the peptic activity was detected at the same distance as that of group I pepsinogen. The immunoreactive pepsinogen I in the urine and serum was then examined by radioimmunoassay. Pepsinogen I was detected in the urine of all the patients who had undergone total gastrectomy, the mean value of urinary pepsinogen I being 32.2 +/- 3.83 ng/ml. In 38 of 40 cases, pepsinogen I was detected in the serum at a mean value of 4.17 +/- 0.51 ng/ml. Moreover, the peptic activities and immunoreactive pepsinogen I levels in the urine correlated well. These results suggested that urinary peptic activities detected after total gastrectomy were due to the pepsinogen I previously believed limited to the gastric mucosa. The extra-gastric production of pepsinogen I was therefore strongly suggested.

The renal metabolism of pepsinogen A and C in man.

Pepsinogen A (PGA) and pepsinogen C (PGC) are almost identical low molecular weight proteins with marked differences in renal handling. PGA is present in large amounts while PGC is almost absent in the urine of healthy subjects. Whether the amount of PGA in the urine represents the total amount of PGA that is extracted, is unknown. We, therefore, assessed the renal metabolism of PGA and PGC by measuring PGA, PGC and creatinine concentrations in the aorta and the right renal vein, and in the urine from patients undergoing elective heart catheterization. The renal extractions of PGA and PGC were not significantly different from the extraction of creatinine: 22%, 18% and 24%, respectively. Sixty-eight percent of PGA and 98% of PGC extracted from the circulation were metabolized by the kidney, and fractional metabolism was closely related to the fractional reabsorption of PGA and PGC from the glomerular filtrate. It is concluded that the kidney metabolizes PGA and PGC. The fractional metabolism of PGA and PGC can be calculated from the fractional reabsorption. Further studies on the renal handling of pepsinogens are warranted as they may provide information on factors affecting renal metabolism of low molecular weight proteins.

Renal handling of pepsinogens A and C in man.

1. Fractional excretions of pepsinogens A and C in the urine were investigated in 21 healthy subjects and in 38 patients with chronic renal insufficiency. In eight of the healthy subjects fractional excretions were measured again after oral administration of omeprazole for 9 days. 2. The mean fractional excretion of pepsinogen A was 27.6% (range 4.4-73.9%) in healthy subjects and remained unchanged after omeprazole administration. In patients with renal failure the mean fractional excretion of pepsinogen A was 37.9% (range 7.0-81.9%). The mean fractional excretion of pepsinogen C was 1.0% (range 0.04-6.8%) in healthy subjects and decreased after omeprazole. In patients with chronic renal diseases a sharp rise in fractional excretion of pepsinogen C was observed once glomerular filtration rate was less than 40 ml/min. 3. Fractional excretion of pepsinogen A was unexpectedly high for a negatively charged protein with a molecule mass of 40,000 daltons. This might be explained by the presence of the positively charged activation peptide. Furthermore, pepsinogen C seemed to be almost entirely reabsorbed from the glomerular filtrate and a tubular reabsorption maximum appeared to be present. Pepsinogen C may, therefore, be a new marker of tubular function. The cause of the remarkable difference in tubular handling of two quite similar low-molecular-mass proteins remains to be elucidated.

Heterogeneity of pepsinogens in the urine of children.

The existence of a significant linear relationship between the concentration of chlorides and the activity of pepsinogen (PG) in the urine was ascertained in the case of full-term infants 3 days to 6 weeks of age. At the age of 4-6 weeks, a significant relationship was found between the urinary pepsinogen activity and the urinary creatinine concentration, and between the urinary pepsinogen activity in the urine and the urine osmolality. Immediately after birth, the Pg7 fraction of PG II in the urine was found in all cases and, at the age of 4-6 weeks, in 11% of cases. In regard to the time factor, the conspicuous drop in the occurrence of the Pg7 fraction corresponds to the new qualitative relationship between the pepsinogen activity in the urine and the creatinine concentration in the urine and between the former and the urine osmolality. In premature infants, the Pg7 fraction disappears more slowly. The spectrum of pepsinogens in the urine was examined in children suffering from various diseases. In a girl with lymphoma, we found the Pg7 fraction, but this finding was temporary only.

Effect of high dose omeprazole on gastric pepsin secretion and serum pepsinogen levels in man.

To investigate the effect of omeprazole on serum and urinary pepsinogens and on gastric pepsin, 8 healthy male volunteers were studied before and after 9 days of treatment with omeprazole 60 mg/day p.o. Fasting serum samples and 24 h urine specimens were obtained, and gastric contents were aspirated at 15-min intervals, 4 prior to and 6 during pentagastrin 1.5 micrograms.kg-1.h-1 i.v. during intra-gastric perfusion with NaCl 0.9% and phenol red 3 mg.ml-1 as an inert recovery marker. Basal and pentagastrin-stimulated volume and acid secretion were significantly decreased. The basal and pentagastrin stimulated pepsin output remained unchanged but pepsin concentration in gastric secretion was increased. Administration of omeprazole resulted in a significant increase in the serum PGA and PGC levels. The 24-h urinary excretion of PGA increased, but that of PGC remained unchanged, and so did the renal clearances of creatinine and pepsinogen A. The renal clearance of pepsinogen C decreased. It was concluded that omeprazole did not affect gastric pepsin output, but, due to the decreased volume output, the concentration of pepsin in the gastric secretion was increased. Omeprazole increased the serum levels of pepsinogen A and C because more pepsinogen was released into the systemic circulation. This might be due to greater back-diffusion of pepsinogen from the gastric mucosa into the systemic circulation as a result of the higher pepsinogen concentration in gastric secretion.

Newly characterized genetic polymorphism of uropepsinogen group A (PGA) using both isoelectric focusing and immunoblotting.

Genetic polymorphism of uropepsinogen group A (PGA) was characterized in human urine using a technique involving both polyacrylamide gel isoelectric focusing and immunoblotting with an anti-PGA antibody. PGA was clearly separable into five fractions, termed I to V in order of decreasing anodal mobility. The most slowly migrating fraction V was composed of F (fast) and/or S (slow) band(s). The population frequencies of the three patterns of fraction V (F, FS, and S) and family studies indicated that PGA V is controlled by a pair of alleles, PGA V*F and PGA V*S, at a single autosomal locus, and that both are codominant. The frequencies of the genes are 0.07 for PGA V*F and 0.93 for PGA V*S.

New detection method for uropepsinogen (PGA) using isoelectric focusing and immunoblotting techniques.

Uropepsinogen (PGA) was isolated and purified from human urine using a column chromatography series. The purified PGA was injected into a rabbit and a PGA-specific antibody was obtained. PGA isozymogen in human urine could be detected reproducibly by immunoblotting using this antibody after isoelectric focusing electrophoresis (IEF) on polyacrylamide gels. This technique may prove to be useful in the genetic study of PGA polymorphism.