Assay for 5-hydroxylysine and L-lysine in human and rat urine and in bone by gas chromatography.

An accurate method for the determination of collagen to study its distribution and turn-over in different tissues is described. 5-Hydroxylysine (5Hylys) is an amino acid that is apparently present in no other protein except collagen and, as it is metabolised only to a minor degree compared with 4-hydroxyproline (4Hypro), it has been suggested as a better marker of the collagen metabolism. Interest in this amino acid has increased recently because the levels of 5Hylys in urine and in different tissues may offer a new basis for detecting pathologies of the collagen molecule. This paper describes a method for the quantitative determination of 5Hylys and lysine (Lys) by gas chromatography (GC) in human and rat urine and in rat bone. The limit of detection was 350 pmol ml-1 for 5Hylys and 200 pmol ml-1 for Lys for all the biological samples. This method therefore provides a complete view of the metabolism of this amino acid and of the tissue it comes from.

A proposal for standardizing urine collections for bone resorption markers measurement.

Diurnal variations in the excretion of bone resorption markers were assessed in order to identify the type of urine collection which provides the most information on bone resorption rate and its relation to measuring bone dynamics in a postmenopausal population. Sixty women, ages 43-67 and without disease or treatment known to affect bone mineral density, were divided into two groups on the basis of femoral mineral density T-score: <1.5 (Group I), >1.5 (Group II). Bone formation was assessed by measuring bone alkaline phosphatase activity and osteocalcin concentration, bone resorption by urinary hydroxyproline, pyridinoline and deoxypiridinoline, N-telopeptide, galactosyl hydroxylysine, and CrossLaps. To identify the more appropriate collection times, urine samples were collected from 7 am to 3 pm; from 3 pm to 11 pm; from 11 pm to 7 am. Twenty-four hour urine collection and first morning void urine samples were also measured. The findings suggest that nocturnal collection and first morning void samples provide the most reliable data on the rate of bone degradation, possibly showing bone loss not only in osteopenic patients but also in women with a low T-score. Nocturnal and first morning samples should therefore be recommended in order to standardize sample collection, as they enable an accurate assessment of bone resorption markers and improved comparability to results from different studies, as well as a less cumbersome collection modality.

Ovariectomy in the rat induces a rapid increase in the urinary excretion of hydroxylysine glycosides and non-reducible crosslink residues.

The ovariectomized rat is the most commonly used animal model of human postmenopausal osteoporosis, exhibiting a high rate of bone turnover with resorption exceeding formation. At present, bone turnover is quantified directly by dynamic histomorphometry. The aim of the present study was to determine whether the measurement of the urinary output of some specific bone collagen catabolites--pyridinolines and hydroxylysine glycosides--could be used to indirectly monitor the initial phase of bone turnover increase in ovariectomized 90-day-old rats. Ninety-day-old female rats were randomly divided into three groups (n = 6): ovariectomized, sham-operated and non-treated controls. Urine samples (24 h) were collected 6 days before surgery and twice weekly for the 4 weeks following ovariectomy. Urinary excretion of pyridinoline (PYD), deoxypyridinoline (DPD), glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) were measured. As expected, ovariectomy was associated with a significant decrease in bone mineral density in both the proximal tibial and distal femoral metaphysis. Compared with both sham-operated and control animals, ovariectomized rats showed significant increases in PYD, GGHYL, and GHYL urinary output 8 days after surgery and in DPD output after 15 days. These changes were maintained throughout the study. The results confirm that measurement of the urinary excretion of pyridinolines and hydroxylysine glycosides represents a powerful tool for detecting the onset of bone turnover in ovariectomized 90-day-old rats.

Longitudinal changes in bone mineral density and bone turnover in postmenopausal women with primary hyperparathyroidism.

The aims of this study were to determine 1) whether primary hyperparathyroidism (PHPT) is associated with accelerated bone loss in postmenopausal women, 2) whether bone mineral density (BMD) and bone turnover change to a similar extent with surgery and hormone replacement therapy (HRT) in these patients, and 3) whether biochemical markers of bone turnover measured at baseline can be used to predict the change in BMD in these patients after different therapies. We studied 33 postmenopausal women with PHPT; their ages at the time of study ranged from 48-80 yr (mean +/- SD, 63 +/- 10). Total body (TB), lumbar spine (LS), and femoral neck (FN) BMD and biochemical markers of bone turnover were measured at baseline and 10-30 months (19 +/- 5) after parathyroid surgery, HRT, or no treatment. BMD was measured in 33 age-matched healthy controls at baseline and at a mean of 24 months. Baseline biochemical markers of bone turnover were measured in controls. In PHPT at baseline, the mean z-score of BMD was -1.25 at TB (95% confidence interval, -1.64 to -0.86), -0.95 at LS (-1.37 to -0.53), and -1.30 at FN (-1.65 to -0.95), whereas the mean z score was 0.45 for serum carboxy-terminal propeptide of human type I procollagen (0.02-0.89), 1.05 for bone alkaline phosphatase (0.38-1.71), 2.38 for 24-h urinary excretion of cross-linked N-terminal telopeptide of type I collagen (NTx; 1.63-3.13), and 2.36 for 24-h urinary excretion of galactosyl hydroxylysine (1.97-2.74). After surgery and HRT, BMD increased and bone turnover decreased during the follow-up. In the untreated group, BMD decreased at TB and FN, and levels of bone alkaline phosphatase, NTx/creatinine, and galactosyl hydroxylysine/creatinine increased. When the rate of change in BMD (percentage per yr) was compared with that in the control group, bone gain was significant at all three skeletal sites after surgery and HRT, and bone loss was significant at TB and FN, but not at LS, in the untreated group. There was a weak, but significant, correlation between baseline urinary NTx and the change in femoral neck BMD in the untreated group (r = -0.36; P = 0.05). We conclude that untreated postmenopausal women with PHPT have low BMD resulting from accelerated bone loss at the TB and FN. Surgery and HRT both restore BMD and bone turnover toward normal in postmenopausal women with PHPT. A single measurement of bone turnover is insufficient to predict BMD changes in individual patients with PHPT.

Comparison between urinary pyridinium cross-links and hydroxylysine glycosides in monitoring the effects of ovariectomy and 17 beta-estradiol replacement in aged rats.

This study was undertaken to assess the sensitivity of hydroxylysylpyridinoline (HP), lysylpyridinoline (LP), galactosylhydroxylysine (GHyl) and glucosylgalactosylhydroxylysine (GGHyl) to monitor bone response to estrogen deficiency and replacement by comparing their excretory patterns in ovariectomized aged (11-14 months old) rats. The ovariectomized (OVX) rats were randomized into two groups: (1) OVX plus vehicle; (2) OVX plus 17 beta-estradiol (17-beta E, 10 micrograms/kg, s.c., 4 days/week). Treatment with 17-beta E started immediately after OVX and continued for 60 days. The collagen catabolites were measured in urine for 1 month before OVX and thereafter for 60 days. In temporal coincidence with urine collection, bone area and bone mineral density (BMD) of lumbar vertebrae, femoral diaphysis and distal metaphysis were measured by dual-energy X-ray absorptiometry. In the untreated rats, BMD of the femoral metaphysis and lumbar vertebrae decreased significantly and the urinary excretion of LP, HP, GHyl and GGHyl increased with different patterns. In the treated rats, 17-beta E replacement prevented the increment in LP excretion, partially prevented the increase in HP excretion, but had no effect on the excretion of GHyl and GGHyl. In conclusion pyridinolines and glycosides have different sensitivities to the bone response to OVX. Glycoside excretion after OVX also reflects metabolic processes not strictly related to bone loss and, in contrast with LP, is not sensitive to estrogen replacement.

Effect of bisphosphonate therapy and parathyroidectomy on the urinary excretion of galactosylhydroxylysine in primary hyperparathyroidism.

BACKGROUND: In patients with mild or asymptomatic primary hyperparathyroidism a reliable index of bone resorption might be useful for appropriate management. Hydroxyproline is the most commonly used marker of bone resorption but its low specificity and sensitivity are known. Galactosylhydroxylysine, an amino acid mainly represented in bone collagen, has been proposed as a more suitable index of bone resorption. In this study we evaluated the sensitivity of galactosylhydroxylysine and hydroxyproline assays in following the changes of their urinary levels in 12 patients with mild primary hyperparathyroidism before and after treatment with bisphosphonate and surgery. METHODS: Serum and fasting urine specimens were obtained from 12 women with mild primary hyperparathyroidism before and after bisphosphonate treatment (2.5 mg daily for 5 days, intravenously) and after a further 25 days; in 7 patients biochemical tests were also performed 1 and 6 days after parathyroidectomy. Galactosylhydroxylysine was assayed by an HPLC method and hydroxyproline by a RIA commercial kit. RESULTS: Baseline galactosylhydroxylysine urinary levels were far above the normal range in all the patients whilst in 8 of them baseline hydroxyproline levels were normal. Bisphosphonate treatment significantly decreased bone turnover as shown by a significant fall in serum calcium (from 2.9 to 2.6 mmol/l; P < 0.001) and in galactosylhydroxylysine and hydroxyproline (-55 and -31% respectively). Twenty-five days after the end of treatment, resorption increased again and serum calcium and galactosylhydroxylysine, but not hydroxyproline, rose significantly towards basal levels. One day after parathyroidectomy serum calcium, galactosylhydroxylysine and PTH showed reduction below normal ranges. PTH and galactosylhydroxylysine returned to normal values at day 6 after parathyroidectomy. No changes in hydroxyproline levels were seen. Galactosylhydroxylysine, but not hydroxyproline, correlated significantly with serum calcium and PTH. CONCLUSION: Galactosylhydroxylysine appears to be a sensitive index of bone resorption, useful in the clinical assessment of bone involvement and in the management of patients with mild primary hyperparathyroidism.

Biochemical markers for detecting bone metastases in patients with breast cancer.

A study was carried out to assess the best use of biochemical bone markers to exclude metastases in patients with breast cancer. Urinary galactosyl-hydroxylysine and serum alkaline phosphatase were used to monitor bone resorption and deposition, respectively. Hydroxyproline was also measured. In a selected population of patients, possibly affected by metastases on the basis of scintigraphic examination, which is highly sensitive but poorly specific, we assessed the efficiency of the markers by a double statistical analysis. In this group, the only marker able to predict metastases was galactosyl-hydroxylysine.

A study of osteoporosis as it relates to metabolic manifestations in edentulous women.

Among some patients, regardless of age, the jaw loses bone mass, leading to loosening and falling out of otherwise healthy teeth. This study seeks to establish whether this bone loss is associated with the metabolic manifestations of other forms of localized decalcifications, such as in Paget's disease, or with generalized osteoporosis. Sixteen women being fitted with dental implants to compensate for bone losses provided 24-hour urine samples for the quantitative determination of calcium and galactosyl hydroxylysine, a bone collagen metabolite. These patients provided demographic information, relevant medical, dental, and dietary history, a profile of their current medications, and the status of their smoking and exercise habits. Urinary excretion of galactosyl hydroxylysine, which is increased in the presence of progressive increased bone resorption, remained within normal values in the patients of this study. These results suggest that the thinning of the jaw bones and subsequent tooth loss of these subjects were osteoporotic processes too limited and too localized to produce measurable increases in urinary bone metabolites.

High predictivity of galactosyl-hydroxylysine in urine as an indicator of bone metastases from breast cancer.

We measured the urinary excretion of galactosyl-hydroxylysine (GH) and hydroxyproline in two groups of women with breast cancer, with (M+, n = 24) and without (Mo, n = 30) clinical, scintigraphic, or radiological evidence of bone metastases. Both these compounds are excreted in larger amounts in the M+ group than in the Mo patients. However, GH, which is a specific marker for bone collagen, provides better predictivity for bone metastases than does hydroxyproline: 92% sensitivity and 90% specificity vs 74% and 79%, respectively, for hydroxyproline.

Collagen metabolite excretion as a predictor of bone- and skin-related complications in spinal cord injury.

Immediately after the trauma, spinal cord injury patients have an increased rate of collagen synthesis and an even greater increase of collagen degradation. Collagen lost from bone is implicated in the etiology of osteoporosis and heterotopic ossification, and collagen lost from skin might lead to a propensity to develop pressure ulcers. The urinary excretion of two collagen metabolites has been monitored and their fluctuation related to the onset of skin or bone complications in these patients. One metabolite, glucosyl-galactosyl hydroxylysine, is more abundant in skin collagen; the other, galactosyl hydroxylysine, is more abundant in bone collagen. The excretion of both metabolites increases after injury, reaching a peak between three and six months after injury, and declines gradually, reaching control values about a year after injury. If a skin pressure ulcer develops, the urinary excretion of the diglycoside remains elevated instead of gradually decreasing. Similarly, if osteoporosis or heterotopic ossification is diagnosed, the monoglycoside excretion does not return to control values until the bone turnover stabilizes. Monitoring of the urinary excretion of both glycosides might prove helpful in prompting early examination to establish the presence of emerging skin and bone complications. Thus, aggressive preventive therapy could be given sooner.

Excretion of urinary collagen metabolites correlates to severity of pulmonary disease in cystic fibrosis.

The urinary excretion of collagen metabolites (hydroxylysine, hydroxyproline and proline) was significantly increased in 10 patients with cystic fibrosis and chronic broncho-pulmonary Pseudomonas aeruginosa infection as compared with 14 age matched controls. The increase was significantly correlated to impaired pulmonary function (FVC and FEV1). The results indicate that urinary collagen metabolites reflect degradation of lung connective tissue and may be an indicator of the severity of pulmonary disease in cystic fibrosis. Parts of the excreted hydroxyproline may be degradation products of elastin.

Increased urinary excretion of collagen metabolites in cadmium-metallothionein nephropathy.

In order to investigate the effect of cadmium-metallothionein (Cd-MT) on renal reabsorption of collagen metabolites, urinary excretion of hydroxylysine (Hyl), glucosyl-galactosyl-Hyl (Glc-Gal-Hyl), galactosyl-Hyl (Gal-Hyl), and hydroxyproline (Hyp), which are unique collagen metabolites, was determined in rats. Administration of Cd-MT resulted in acute renal failure in the form of proteinuria, aminoaciduria and glycosuria. Protein content in urine was greatly increased 1 day after injection of Cd-MT and decreased from 5 days, while the maximum levels of excretion of amino acids and glucose were observed at 6 days post-injection. The urinary excretion of total Hyp and Hyl, including Glc-Gal-Hyl, Gal-Hyl and free Hyl, were significantly increased at 3, 6 and 8 days after injection of Cd-MT with the maximum level at 6 days. Moreover, the molar ratio of Glc-Gal-Hyl/Gal-Hyl of urine in the Cd-MT-treated group was almost the same as that in the controls. These results suggest that a portion of Hyp, Hyl and its glycosides is normally reabsorbed from the renal tubule in the controls, and Cd-MT exposure caused an increase in urinary excretions of Hyp and Hyl, including its glycosides, through a renal tubular defect in reabsorption of Hyl in the same manner as with common amino acids.

Hereditary nephritis and hypoplastic dysplastic nephropathy: hydroxylysine glycoside excretion and the glomerular basement membrane.

Eleven children aged 5 to 15 years with hereditary nephritis and twelve children with hypoplastic dysplastic nephropathy were studied. The children with hereditary nephritis were divided into 2 groups while correlating the level of renal excretion of hydroxylysine glycosides (HOLG) with ultrastructure of glomerular basement membrane. The first group was characterized by decreased urinary excretion of HOLG and extremely thin basement membranes, the second one - by increased HOLG excretion and thickened membranes, The children with hypoplastic dysplastic nephropathy which were divided into 2 groups according to HOLG excretion differed in presence or absence of anatomic anomalies and in the number of connective tissue dysembryogenetic stigmata. The data are suggestive of an important role and diversity of metabolic derangement of the collagenic component of basement membranes in the pathogenesis of the diseases studied.

Excretion and synthesis of basement membrane disaccharide units in Masugi nephritis.

During nephrotoxic nephritis in the rat, an increased urinary excretion of glucosyl-galactosyl hydroxylysine and of galactosyl-hydroxylysine has been observed in the autologous phase of the disease. This due mainly to an elevation of the polypeptide-bound fraction of these hydroxylysyl glucosides with a molecular weight over 1,000 daltons. The levels of both urinary hydroxylysyl glucosides were correlated with proteinuria. Their increased excretion appears to originate in the lysed glomerular basement membrane. At the same stage of nephrotoxic nephritis, an increased glucosyl transferase activity could be demonstrated in the isolated glomeruli, correlated with albuminuria, attesting a higher turn-over of the disaccharide units of the glomerular basement membrane.